Molluscan Shell Proteins: Primary Structure, Origin, and Evolution

6 ____________________________________________________________________________

Molluscan Shell Proteins: Primary Structure,Origin, and Evolution

Frederic Marin,* Gilles Luquet,* Benjamin Marie,* andDavorin Medakovic{

*UMR CNRS 5561 ‘Biogeosciences,’ Universite de Bourgogne

6 Boulevard Gabriel, 21000 DIJON, France{Center for Marine Research Rovinj, Ruder Boskovic Institute

5 Giordano Paliaga, 52210 ROVINJ, Croatia

I. I

Curre

Copy

ntroduction: The Shell, a Biologically Controlled Mineralization

II. M
olluscan Shell Formation: Developmental Aspects
A
. T
nt To

right

he Larval Shell

B
. T he Juvenile and Adult Shell
C
. T ransient Amorphous Calcium Carbonate
III. T
he Topographic Models of Shell Mineralization
A
. E arly Nacre Descriptions and Models
B
. R ecent Nacre Models and Evolving Views
C
. P rism Models
IV. M
olluscan Shell Proteins: Characterization of Their Primary Structure
A
. E xtremely Acidic Shell Proteins
B
. M oderately Acidic Shell Proteins
C
. B asic Shell Proteins
D
. P artially Characterized Shell Proteins
E
. O ther Molluscan Proteins: The Extrapallial Fluid and the Mantle
F
. R emarks on Molluscan Shell Proteins
V. O
rigin and Evolution of Molluscan Shell Proteins
A
. T he Cambrian Origin of Mollusk Shell Mineralization
B
. T he ‘‘Ancient Heritage’’ Scenario
C
. T he ‘‘Recent Heritage and Fast Evolution’’ Scenario
D
. L ong‐Term Evolution of Shell Matrices and Microstructures:
The Bivalve Example

VI. C
oncluding Remarks
A
cknowledgments
R
eferences
In the last few years, the field of molluscan biomineralization has known a

tremendous mutation, regarding fundamental concepts on biomineralization

regulation as well as regarding the methods of investigation. The most recent

advances deal more particularly with the structure of shell biominerals at

nanoscale and the identification of an increasing number of shell matrix protein

components. Although the matrix is quantitatively a minor constituent in the

pics in Developmental Biology, Vol. 80 0070-2153/08 $35.002008, Elsevier Inc. All rights reserved. 209 DOI: 10.1016/S0070-2153(07)80006-8

210 Marin et al.

shell of mollusks (less than 5% w/w), it is, however, the major component that

controls diVerent aspects of the shell formation processes: synthesis of transient

amorphous minerals and evolution to crystalline phases, choice of the calcium

carbonate polymorph (calcite vs aragonite), organization of crystallites in

complex shell textures (microstructures).

Until recently, the classical paradigm in molluscan shell biomineralization

was to consider that the control of shell synthesis was performed primarily

by two antagonistic mechanisms: crystal nucleation and growth inhibition.

New concepts and emerging models try now to translate a more complex

reality, which is remarkably illustrated by the wide variety of shell proteins,

characterized since themid‐1990s, and described in this chapter. These proteinscover a broad spectrum of pI, from very acidic to very basic. The primary

structure of a number of them is composedof diVerentmodules, suggesting that

these proteins are multifunctional. Some of them exhibit enzymatic activities.

Others may be involved in cell signaling. The oldness of shell proteins is

discussed, in relation with the Cambrian appearance of the mollusks as a

mineralizing phylum and with the Phanerozoic evolution of this group.

Nowadays, the extracellular calcifying shell matrix appears as a whole

integrated system, which regulates protein–mineral and protein–protein inter-

actions as well as feedback interactions between the biominerals and the

calcifying epithelium that synthesized them. Consequently, the molluscan

shell matrix may be a source of bioactive molecules that would oVer interestingperspectives in biomaterials and biomedical fields. � 2008, Elsevier Inc.

I. Introduction: The Shell, a BiologicallyControlled Mineralization

Biomineralization refers to the dynamic physiological process by which a living

organism elaborates a mineralized structure. Biomineralization refers also to

the final product, the mineralized structure, whatever it is, a rigid skeleton or a

nonskeletal mineralization (Lowenstam and Weiner, 1989). In living systems,

biominerals display a wide range of functions: tissues support, UV protection,

shelter against predation, nutrition, reproduction, gravity, light or magnetic

field perceptions, storage of mineral ions (Simkiss and Wilbur, 1989). In the

metazoan world, calcium carbonate skeletons are the most commonly encoun-

tered biomineralizations, and the most abundant, from diploblastic animals,

sponges, and corals to deuterostomes, echinoderms, and chordates. Among

mollusks, calcium carbonate biomineralization exhibits a huge diversity of

morphologies (Lowenstam and Weiner, 1989): epithelial spicules, scales and

plates, operculum, intracellular detoxifying granules, egg capsules, love dart,

pearls, statoconia, and statoliths, but the most well‐known molluscan calcium

carbonate biomineralization is the shell, the primary function of which is to

6. Molluscan Shell Proteins 211

support these soft‐bodied organisms and protect them from predation and

desiccation.

The molluscan shell is an organo‐mineral composite, where the dominant

mineral—aragonite, or calcite, or in particular cases, vaterite—is intimately

associated to an organic matrix, which accounts only for 0.1–5% of the shell

weight. This matrix represents amalgamate of proteins, glycoproteins, chitin

and acidic polysaccharides, secreted by the calcifying tissues during skeleto-

genesis. This mixture is consequently sealed within the skeleton during its

growth. At macroscopic level, the adjunction of organic components to a

mineralized structure enhances the mechanical properties to the whole

organo‐mineral assembly. At molecular level, the matrix plays a key role in

the mineralization process.

According to the terminology introduced by Stephen Mann (1983), the

construction of the shell is the archetype of a biologically controlled miner-

alization. This concept can be summarized by five identification criteria.

(1) The process requires specialized cellular machinery, which means that

the minerals formed are not just by‐products of the metabolic activity but

correspond to a specialized metabolic pathway. (2) The mineral synthesis is

an active process, that is, theminerals are synthesized far from the equilibrium

with the environment. (3) The formed minerals are diVerent in their shape

and size from their inorganically formed counterpart. (4) The minerals are

not formed in direct contact with the environment, that is, the organism has

developed a strategy for delimiting the space where the minerals are synthe-

sized. (5) The biomineralization process is mediated by an extracellular

organic matrix. The molluscan shell complies with all these criteria.

During decades, the molluscan shell matrix was considered as a single

entity and there were considerable eVorts to propose hypotheses on its

putative functions (Bevelander and Nakahara, 1969; Krampitz et al., 1976;

Lowenstam and Weiner, 1989; Simkiss and Wilbur, 1989; for a review, see

Marin and Luquet, 2004). Although these functions are generally accepted,

they have been mainly deduced from detailed micro‐ and ultrastructural

observations of the final product (SEM, TEM), from physical measurements

(XRD), from biochemical characterizations, and/or from in vitro tests that

poorly mimic the real conditions. Until now, a large part of the shell biomin-

eralization process, that is, the mysterious transition from precursor fluids to

the final product, the solid shell, still escapes our comprehension, and the

self‐assembling capacity of shell matrices remains a ‘‘black box.’’ In spite of

our ignorance in knowing each step of the secretory sequence that leads to

the shell, several putative functions are attributed to the associated matrix, as

listed as follows: the shell matrix presumably concentrates locally the pre-

cursors ions; it constitutes a tridimensional framework, acts as a template for

crystals, and allows the nucleation of crystals only where appropriate; it

selects the calcium carbonate polymorph; it controls crystal elongation in

privileged crystallographic axes and inhibits crystal growth by poisoning

212 Marin et al.

their faces; it determines the spatial arrangement of crystal units at diVerentscales, from nanometer tomillimeter. Beside these physicochemical aspects of

matrix–mineral interactions, the organic matrix is likely to display enzymatic

functions and to be involved in cell signaling.

Proteins and glycoproteins represent essential components of the shell

matrix, and there are at least three good reasons for studying these macro-

molecular components from a fundamental point of view. First, obtaining

the structure of shell proteins may allow a better understanding of their

respective functions and may help to refine the biomineralization model.

This in turn may provide solid cues for studying the phylogenetic relation-

ships of the diVerent mineralized tissues and for understanding how these

systems were formed. At last, because the shell is a closed system, which

sooner or later becomes an integral part of the fossil record, knowledge on

the biochemical properties of shell proteins may help to trace their diagenetic

evolution during burial and fossilization and, more generally, may provide a

strong basis for analyzing the diagenesis of skeletal carbonates.

Other reasons for studying molluscan shell proteins lie in the fascinating

and challenging applied perspectives that these proteins oVer (Mann, 2001;

Marin et al., 2007). For example, shell proteins may be employed in nano-

technologies, for micromanipulating nanocrystals and semiconductors. They

can be used for biomimicry purposes, that is, the synthesis at room temperature

of composite materials, which exhibit high‐mechanical properties. Molluscan

shell proteins may also be used as natural bioactive factors, in particular in

bone surgery. The best example is provided by the osteoinductive/osteogenic

properties of nacre matrix and nacre implants. Another domain, which

may benefit from advances in the knowledge on shell proteins, is the pearl

industry, a major economical activity in the South Pacific area. At last, mollus-

can shell proteins may be used as natural biodegradable antiscaling and

antifouling agents.

The aim of this chapter is to review our present knowledge on molluscan

shell proteins and to resituate them in an evolutionary framework. However,

in order to bring a dynamic view of the system, we will first describe some

developmental aspects of the shell and present evolving views on the models

of molluscan mineralization.

II. Molluscan Shell Formation: Developmental Aspects

A. The Larval Shell

A review on the diVerent modes of embryonic and postembryonic mollusk

development is far beyond the scope of this chapter, and we advise the

reader to refer to the very detailed review of Nielsen (2004) for the early

A B

60 µm 70 µm

Figure 1 SEM picture of veliger larvae of M. galloprovincialis. The soft tissues were removed

and the shells were cleaned with dilute NaOCl (0.026% active chlorine, 1 hour). (A) Larval shell

in the later « D » stage. White arrows indicate prodissoconch I/II boundary, which marks the

moment when the valves hermetically enclose larval body. The valves are equal in convexity and

dimension of prodissoconch I is on the order of 80–90 mm. The surface of this layer is character-

ized by nearly smooth to small ‘‘pitted–furrowed’’ zones with faint radial striations. Prodisso-

conch II is distinctly co‐marginally striated. On the ventral side of the well‐calcified left shell

valve (black arrow), linear prodissoconch I hinge is visible. (B) The inner part of prodissoconch I

hinge is simple and still not interlocking the valves. At the start of prodissoconch II development,

the central part of the hinge is disconnected. The valves fit together by tiny rugosities, starting

denticulations on the edge of hinge (black arrows). From this primordial structure during further

larval development, hinge teeth and ligament, umbo of the shell will be formed. Calcified portion

contains tiny unequal granules, which become smaller toward the prodissoconch I/II boundary.


developmental stages. Let us however recall few general considerations about

mollusk development and the phylogenetic position of the group. Mollusks

are triploblastic protostomial (the blastopore gives the mouth of the adult)

schizocoelomates (the coelomic cavity is produced by the splitting of the

mesoderm). Within protostomes, mollusks belong to the lophotrochozoan

superphylum (Aguinaldo et al., 1998; Halanych et al., 1995), together with

brachiopods, bryozoans, annelids, platyhelminthes, acanthocephala, and

some minor phyla. They are eutrochozoans, the characteristic of which is

to produce a swimming‐ciliated larva, the trochophore larva (Lecointre and

Le Guyader, 2001). In the early embryonic stages, the mollusk development

exhibits several similarities with that of annelids, a trait that brings these two

phyla in a close phylogenetic relationship (Nielsen, 2004).

Contrarily to vertebrate or sea urchin eggs, the molluscan egg undergoes a

determinate spiral cleavage (except for cephalopods), which knows several

variations from species to species. The first cleavages are, for a majority of

species, unequal, and one of the cells produced after the second division, the

D cell, is bigger than the three others. The subsequent unequal divisions of

the D cell produce the 2d micromere, which will give the cells that produce

214 Marin et al.

the shell. This general scheme knows some exceptions: for the gastropod

Patella, the shell gland develops mainly from 2a and 2c micromeres (Dictus

and Damen, 1997). It is striking to notice that, in gastropods, the sense of the

third cleavage (the first spiral cleavage, right‐ or left‐handed) determines

the sense of the shell coiling, but the link between the two remains obscure.

The shell coiling seems under the control of a recessive gene called sinistral

(Schilthuizen and Davison, 2005).

In mollusks, two types of postembryonic development are observed

(Martoja, 1995). First, an indirect development is shared by most of mollus-

can classes (in particular bivalves and gastropods) and characterized by a

transition from a ciliated trochophore to a veliger larva and a metamorphosis

from the veliger to a juvenile. The first transition implies the acquisition of

a velum used for swimming. The veliger larval stage is typical of mollusks.

In some cases, like in the freshwater unionid bivalves, the veliger larva, called

glochidium, adopts a parasitic life mode on fish gills. In gastropods, the

veliger stage corresponds to the larval torsion, which twists the head and

foot by 180� relative to the shell, mantle, and visceral mass. The main

metamorphosis occurs when the pelagic veliger larva settles down for a

benthic existence. This transformation is profound and corresponds to the

disappearance of the velum, the development of the foot, and the organiza-

tion of the digestive gland and of the reproductive organs (Bonar, 1976). The

second mode of development is direct, without larval stages neither meta-

morphosis, which implies that juveniles look like adults in reduction. This

most derived developmental mode is particular of cephalopods and is char-

acterized for most of them, exceptNautilus, by an internalization of the shell.

The following description is mainly applied to bivalves and gastropods, the

two main classes by their number of species.

The first steps of the larval shell formation occur during the trochophore

stage. The shell—of ectodermic origin—is produced by a group of cells

located on the posterior side of the larva. These cells define the shell field

(Kniprath, 1981). The shell field is diVerentiated at the end of the gastrulation

stage, by the thickening of the median portion of the ectoderm in the post‐trochal dorsal region (Moor, 1983). Cells of the shell field invaginate, accord-

ing to various pathways described by Kniprath (1981), and this invagination

produces the transitory shell gland, also called preconchylian gland. Accord-

ing to Kniprath (1977, 1980), the invagination is functionally required, for

allowing the cells at the periphery of the shell gland (cells that are not

internalized during invagination) to produce the early organic membrane,

which will be the first template for calcium carbonate minerals deposition.

This organic lamella is the future periostracum. Let us remind that the

periostracum is the leathery outer layer of the shell, particularly visible in

species like the edible mussel, Mytilus edulis. In the next step, the shell gland

gradually flattens and/or evaginates and spreads by mitotic divisions, while


transforming into the larval mantle epithelium. In the meantime, the early

periostracum expands. Between the periostracum and the cells of the shell

field, the primary mineralization takes place. In bivalves, the first shell,

secreted during the trochophore stage, is called prodissoconch I and is

characterized by a granular aspect (Mao Che et al., 2001). During the

transition from trochophore to veliger stage, the prodissoconch II shell is

secret ed and marks a chang e in the secreto ry regime (Fig. 1). The prodiss o-

conch II stage is indeed characterized by the appearance of growth lines on

the valves. It is suggested that the cells that secrete the prodissoconch II shell

are diVerent from those which produce the prodissoconch I (Mao Che et al.,

2001). Following metamorphosis of the veliger larva, the dissoconch shell is

formed. Again, this change is marked on the outer surface of the shell

by an accentuated growth line. As mentioned by Jablonski and Lutz

(1980), the terminology used for describing the successive shell stages in

gastropods is diVerent: the first shell formed at late trochophore stage is

then called protoconch I, the second shell secreted during the veliger larval

stage, protoconch II, and the postmetamorphosis shell, teleoconch.

In comparison to other phyla, in particular echinoderms and arthropods,

the connection between the physiology of the larval shell development and

the underlying genetic machinery, which controls and patterns the process, is

supported by a limited number of studies in mollusks. Of outstanding interest

are the works of Moshel et al. (1998), Jacobs et al. (2000), Wanninger and

Haszprunar (2001), Klerkx et al. (2001), Nederbragt et al. (2002), and

Hinman et al. (2003). In particular, the first four cited papers underline the

key role played by engrailed (En) in molluscan shell development. The En

class encodes homeodomain‐containing DNA‐binding proteins involved in

major steps ofmetazoan development (Hidalgo, 1996). These multifunctional

transcription factors are, among others, involved in the patterning of the

nervous system (neurogenesis); in the body segmentation in annelids, arthro-

pods, and vertebrates; and in several other derived functions, such as the

specification of the ventral compartment in vertebrate limbs, or the pattern-

ing of the mid‐hindbrain boundary (Gibert, 2002). Besides working at tran-

scriptional level, En also modulates translation and seems to be able to act as

morphogens (Morgan, 2006). En has been identified in most metazoan

lineages, including mollusks (Wray et al., 1995). In the marine mud snail

(Ilyanassa) embryo, the expression of En is localized only in the shell gland

(Moshel et al., 1998). In the trochophore larva of the tusk shell Antalis, En is

expressed in shell‐secreting cells at the border of the protoconch. However,

after metamorphosis, En expression was not observed in the cells that pro-

duce the adult shell, the teleoconch (Wanninger and Haszprunar, 2001). In

the chiton, En is expressed in region that bound skeletal plates, and in the

clam, En expression surrounds each developing valve and the hinge (Jacobs

et al., 2000). According to these authors, En would be directly involved in

216 Marin et al.

skeletogenesis by marking the skeletal boundaries. They also claim that En

would display the same function in other calcifying bilaterian metazoans.

This suggests that the acquisition of a calcified skeleton may be a unique

event across metazoan phylogeny, which would explain the sudden emer-

gence of calcified skeletons at the dawn of the Cambrian times. This view on

the direct role of En in skeletogenesis was contested by Nederbragt et al.

(2002), for whom En is primarily involved in delimitating compartment

boundaries between cells of the shell gland and the other ectodermic cells.

If so, the contribution of En to shell formation is undirect. The paper of

Hinman et al. (2003) underlines the major role of Hox1 and Hox4 during

the development of the abalone larva. In the trochophore larva, Hox1 is

expressed in a ring of cells corresponding to the outer mantle edge. Hox4

is expressed in the mantle, but at later stage, after the larval shell is fully

formed. Hinman et al. suggest that both Hox genes may have been co‐optedinto a role in patterning shell. At last, some developmental genes may also

indirectly contribute to the shell formation, by their absence of expression in

the shell‐forming cells. This is the case for E32, a gene encoding a putative

RNA‐binding protein, not expressed in the shell gland of Patella, but

expressed in the cells, which are maintained in an undiVerentiated state

(Klerkx et al., 2001). E32 would block the cell diVerentiation process. Clearly,

further studies, as the ones described here, are required for mollusk phylum

before a complete picture of the role of developmental genes in shell formation

can emerge.

Another important aspect in the formation of the embryonic shell deals

with the enzymatic activity that occurs during the whole process of larval

development. This aspect has, however, been widely neglected. In the fresh-

water snail, Lymanea, the old study of Timmermans (1969) showed that the

expression of alkaline phosphatase (ALP) was the highest during the evagina-

tion process, while the expressions of DOPA‐oxidase (tyrosinase) and peroxi-

dase were maximal at the borders of the shell gland, after evagination. This

zone corresponds to the zone where the periostracum is secreted. In the edible

mussel, Mytilus, the level of carbonic anhydrase was recorded during

the whole developmental process. In larvae, high expressions of carbonic

anhydrase were found to precede the formation of the shell field in

the gastrula stage, the formation of the shell gland and periostracum in the

trochophore stage, and the mineral deposition in the prodissoconch I and

prodissoconch II stages (Medakovic, 2000). In the embryo of the freshwater

snail Biomphalaria glabrata, the temporal and spatial activities of ALP,

peroxidase, and acid phosphatase were analyzed by histochemical staining

(Marx en et al ., 2003b ,c). An ALP acti vity was observed in trocho phore larva

in the invaginated shell field (shell gland), prior secretion of any shell material.

A peroxidase activity was found in small vesicles of cells involved in the

secretion of the periostracum. Acid phosphatase was localized in the shell


field and around the shell field invagination. At last, a recent key paper ofWeiss

et al. (2006) underlined the importance of chitin synthase, a transmembrane

glycosyltransferase, which synthesizes chitin. In situ hybridization experiments

performed on larvae of the mussel Mytilus galloprovincialis showed that the

chitin synthase transcript was present in early and late veliger stages, in the cells

in close contact with the larval shell. A striking finding is the presence of a

myosin motor head domain in the intracellular N‐terminus of the identified

chitin synthase. This clearly suggests, for the first time, that the cytoskeleton

plays a crucial, although poorly understood, role in chitin formation.

B. The Juvenile and Adult Shell

Once the metamorphosis of the veliger has occurred, the resulting juvenile

mollusk calcifies rapidly, and its shell growth approximately follows a von

BertalanVy law (Seed, 1980), during the whole life of the animal. Classically,

the physiology of molluscan shell calcification can be described as a succes-

sion of compartments (Wilbur and Saleuddin, 1983), where the central ele-

ment is the mantle, the thin organ, which coats the inner surface of the shell,

the other compartments being the extrapallial space and the shell. The mantle

is a polarized tissue, and comprises an inner epithelium, in contact with the

ambient medium (e.g., seawater), the mantle interior, which comprises pallial

muscles, connective tissues, nerve fibers, and finally the outer epithelium. As

shown in Fig. 2, the outer epithelium is the epithelium, which mineralizes the

shell. Whether this epithelium is in direct contact with the shell is still debated

(see below).

The extremity of the mantle is characterized by a succession of folds, three

among bivalves two among gastropods. The ridge between the outer and

median folds defines the periostracal groove, which secretes the periostracum.

As described in Section II.A, the primary role of the periostracum is to

provide a support for the mineralization. Its second important role is to

delimitate and seal the space where the mineralization takes place. Actually,

the invention of the periostracum corresponds to an old strategy that mol-

lusks have set up for mineralizing in a confined space, the extrapallial space.

The periostracum is secreted as a liquid film of tyrosine‐rich precursors, whichrapidly becomes insoluble and sclerotized by a quinone‐tanning process

(Waite, 1983).

Precursor ions—calcium and bicarbonate—are taken up from the ambient

medium, through the inner epithelium, or from the gill; both ions can also

originate from the mollusk metabolism (food and fluids). They transit in the

connective tissues of the mantle via the hemolymph—the interstitial fluid

that bathes the mollusk cells—and are directed toward the outer mantle

epithelium. Calcium and bicarbonate ions can be actively extruded from

Inner epithelium

Outerepithelium

Mucocyte

Pallial muscle

Periostracum

Periostracal groove

Extrapallial space

Nacreouslayer

Prismaticlayer

Figure 2 Physiology of the shell calcification in a nacro‐prismatic bivalve, redrawn from

Saleuddin and Petit (1983). The calcification takes place at the distal border of the shell in the

extrapallial space. In the outer epithelium, the cells responsible for the deposition of nacre are

not localized in the same area as the ones responsible for the secretion of prism precursors.

The prisms and nacre tablets are not drawn to scale.

218 Marin et al.

the cytosol to the second compartment involved in shell formation, the extra-

pallial space (Fig. 2). However, in several cases, calcium can be temporarily

stored as intracellular or extracellular amorphous granules. This has been

particularly well studied for bivalves (Fournie and Chetail, 1982; Istin, 1970;

Istin and Girard, 1970) for which a colocalization of granules and carbonic

anhydrase was observed. Amorphous granules can be used by the cells for

detoxifying the cytosol from an excess of calcium or heavy metals (Simkiss,

1977, 1993).Granules are also a source of calcium,which is rapidly available, in

particular, for a rapid shell mineralization and repair. The possibility of outer

epithelial cells to release intracellular calcium granules by exocytosis is not

documented, although suspected.

The second—already mentioned—compartment is the extrapallial space

(Fig. 2). This space is supposed to concentrate the precursor mineral ions,

owing to calcium and bicarbonate pumps. However, these pumps have not


been localized on the outer epithelium, mainly because they have been poorly

characterized at the molecular level (Endo et al., 2003). The extrapallial space

is supposed to be the place where the ‘‘mysterious’’ transition from the liquid

precursors to the solid biominerals occurs. The concept of self‐assembling is

currently put forward for explaining how the shell matrix and the mineral

ions interact in a controlled manner to produce a finely structured organo‐mineral material. Chemical analyses of the extrapallial fluid have shown that

it is supersaturated with respect to calcium carbonate (Misogianes and

Chasteen, 1979; Moura et al., 2000). This fluid is also enriched in mineral/

organic mineralization inhibitors, without which calcium carbonate would

precipitate anarchically. This obviously never happens.

The extrapallial space is also the place where the shell matrix is secreted

and where the subtle transition from liquid to solid operates. As said in the

introduction, and detailed later, this matrix is a mixture of proteins, glyco-

proteins, acidic polysaccharides, chitin, and presumably lipids. The matrix

interacts with the mineral ions and controls the shape of the produced

crystals. It is consumed by the system, that is, ‘‘entrapped’’ within the

construction in progress. This means that it has to be brought to the miner-

alization front when required during the whole mineralization period. It is

also likely that several proteins, which are present in the extrapallial fluid,

are not incorporated in the shell. Another unknown parameter is the precise

temporal sequence of secretion of the shell matrix. On the other hand, we

know that the secretory regime is diVerent depending on the position of

the cells involved in this process on the outer epithelium. This has been

demonstrated, both at transcriptional and protein levels, in particular for

species, which exhibit a bitextured shell (an outer prismatic layer, an inner

nacreous layer; Fig. 2). The outer epithelial cells that secrete the matrix

involved in controlling the prism formation occupy a more distal position

(from the shell hinge) than the ones that secrete the matrix involved in the

nacre deposition (Jolly et al., 2004; Sudo et al., 1997). A paper from Takeuchi

and Endo (2005) confirms the existence of two zones in the outer mantle

epithelium, with transcripts specific of the prism zone, transcripts specific of

the nacre zone, and transcripts present in both. Attempts to introduce

an epithelial cell typology have been made (Sud, 2002). At last, because

the deposition of calcium carbonate is accompanied by the release of pro-

tons, these latter have to be reabsorbed by the calcifying epithelium, for

precluding acidification of the extrapallial fluid, and possible resolubilization

of the newly formed minerals. It has been suggested that proton pumps are

involved in extruding protons from the extrapallial fluid toward the cytosol.

However, this process is poorly documented for mollusks (Coimbra et al.,

1988). It has also been suggested that proton pumps work, in some cases,

in the reverse sense, inducing then acidosis in the extrapallial space (Moura

et al., 2003).

220 Marin et al.

The classical view described here above seems a priori ‘‘well established.’’

However, several ‘‘evidences,’’ like the transit of precursor ions, are indirect

or deduced from the general metabolism of the calcifying mantle. Several

physiological aspects are left in the dark or bring unsatisfactory answer. For

example, our description attributes a key role to the extrapallial fluid. This

suggests that there are no direct contacts between the secreting epithelium

and the location where mineralization occurs. This view of a ‘‘remote control

of the mineralization by the epithelium’’ is questionable (Addadi et al., 2006).

Some authors consider that the mantle cells have to keep close contact with

the mineralization front in order to receive feedback molecular signals from

the newly formed biominerals.

Another point, which remains obscure, is the function of hemocytes—free

circulating cells of the hemolymph—in the shell construction process. Hemo-

cytes play a major role in the immune defense of mollusks (Glinski and

Jarosz, 1997) and in tissue repair (Serpentini et al., 2000). Their role in shell

repair processes has already been underlined (Bubel et al., 1977; Watabe,

1983), but their contribution to the normal shell formation may be widely

underestimated. A milestone paper from Mount et al. (2004) attributes to

hemocytes (of the granulocyte type) of the oyster Crassostrea virginica, an

important function in the normal shell construction process, by releasing

calcite crystals—not amorphous granules—which can be remodeled at the

mineralization site. So far, it is diYcult to evaluate whether this process is

particular to the studied model or whether it is a general metabolic pathway

that mollusks use for mineralizing their shell.

C. Transient Amorphous Calcium Carbonate

One aspect, which has for a long time been passed largely unnoticed, but

which needs to be urgently reevaluated, is the key role played by amorphous

calcium carbonate (ACC) in molluscan shell formation and, more generally,

in all calcium carbonate biomineralizations (Weiner et al., 2003). A mineral

phase is considered to be amorphous when it lacks the long‐range order,

that is, when it does not have the regular repeating lattice structure that

provides the basis for so many of the features of a crystal (Simkiss, 1993).

Several amorphous minerals can exhibit a short‐range order, which is not

conserved at a longer scale. This implies that the mineral does not give an

X‐ray diVraction pattern characterized by spots materializing the diVerentdiVraction plans. Amorphous minerals display several advantages in com-

parison to their crystalline equivalent: their formation requires less energy;

they are more easily solubilized, which means that their constitutive ions

are more easily available; they incorporate ionic impurities with a higher

tolerance than the crystalline form; they exhibit percolation channel that


allows ion di Vusion, that is, they can be more easily remod eled ( Simkiss,

1991 ).

Recent X ‐ ray di Vracti on data indica te that the early larva l shell miner ali-

zatio n (prod issocon ch I) may be a transient a morphous phase, a fact that

earlier works on Tridacn a squamo sa ( LaBarb era, 1974 ) or on Crassos trea

gigas (Lee , 1990 ) had not detected . The presence of ACC has been de mon-

strated for the fres hwater snail, B. glabr ata ( Hass e et al ., 2000 ; Marxen et al.,

2003a) , for which the trans form ation of ACC into aragoni te could be

recorded at diV erent developm ental stage s. In the musse l, M. eduli s , the

first mineral formed is also ACC, but its quantity dramatically drops only

40 hours after fertilization (Medakovic, 2000), in profit of the aragonitic

phase. The early developmental stages of the oyster, Ostrea edulis, are also

marked by an extremely broad X‐ray diVraction ‘‘peak’’ that may be inter-

preted as ACC (Medakovic et al., 1997). In the clam Mercenaria mercenaria,

the prodissoconch I contains ACC, which transforms after several days in

aragonite. In the oyster C. gigas, the prodissoconch I contains ACC and

aragonite (Weiss et al., 2002). By extrapolating these data to adult shells, and

by keeping in mind the presence of amorphous granules in the molluscan

mantle tissues, it is conceivable that the whole adult shell formation occurs

via a transient ACC phase. As we mention in the next section, recent findings

have shown that interfacial amorphous phases exist also in the ‘‘finished’’

shell, which suggests a stabilization mechanism of ACC.

III. The Topographic Models of Shell Mineralization

One key issue in research on molluscan shell biomineralization is the under-

standing of the relationships between the organic matrix and the mineral

phase at ultrastructural level. This question is central to current hypotheses

on biologically controlled mineralizations, but is still extremely debated.

From the late 1960s when the early topographic models of molluscan miner-

alization emerged to now, there has been a considerable evolution of the

concepts, partly because of the evolving methodologies used for observing

biominerals. In the early years, scanning electron microscopy (SEM) was the

unique investigation tool. The use of surface etching treatments, partial

decalcification and fixation (Mutvei, 1979), enzymatic degradation brought

additional information by revealing extremely fine substructures (Cuif et al.,

1983). At high magnification, transmission electron microscope (TEM) and,

more recently, cryo‐TEM and atomic force microscope (AFM) brought

surprising ultrastructural informations such as the presence of crystal nano-

domains. Finally, in the last years, XANES (X‐ray absorption near edge

spectroscopy) and NanoSIMS brought additional structural informations.

SEM combined with immunogold proved to be a promising technique (Marin

222 Marin et al.

et al., 2007) for visualizing single‐protein components within biominerals.

In the next section paragraph, we will discuss some recent outcomes on nacre

and prisms, the two most familiar molluscan shell textures.

A. Early Nacre Descriptions and Models

For several reasons such as its economical interest or its mechanical proper-

ties, nacre, also called mother‐of‐pearl, is still one of the most‐studied shell

textures. Many consider it as the reference model because of its apparent

geometrical simplicity and because of its universality among mollusks. Nacre

is indeed a widespread molluscan shell texture, and is, with one exception, a

bryozoan (Weedon and Taylor, 1995), typical of this phylum. It is repre-

sented within the three main classes of mollusks, bivalves, gastropods,

and cephalopods. The ‘‘nacre’’ terminology refers to a well‐defined type of

microstructure, described as follows: small flat tablets of aragonite, about

half a micron thick, which are tightly packed together by organic cement.

The tablets can be rectangular, hexagonal, or rounded, and look like mono-

crystals. They form superimposed layers of uniform thickness. Basically,

there are two broad types of nacre, depending on the disposition of tablets

(Erben, 1972; Nakahara, 1991): ‘‘brick wall’’ nacre, which is classical among

bivalves, ‘‘columnar’’ nacre, found in gastropods (Fig. 3). In the first type, in

cross section, crystals are positioned in staggered rows, just like bricks in a

wall (Checa and Rodriguez‐Navarro, 2005; Oaki and Imai, 2005). Bivalve

nacre tablets have their a, b, and c axes co‐oriented, with the c axis perpen-

dicular to the nacre surface, and the b axis parallel to the local growth

direction of the shell margin (Checa et al., 2006). In the second type, flat

tablets are aligned on top of each other, and thus, form piles (or towers)

of crystals (Lin and Meyers, 2005). Tablets of the same pile are co‐oriented(c axis along the axis of the pile), but from pile to pile, the a and b axes are not

ordered.

TEMstudies have shown that a thin layer of organicmatrix, the interlamellar

matrix, delimitates the lower and upper tablet surfaces (Fig. 3). The thickness of

this matrix is about 20 nm. Within a same lamella, an organic matrix [the

intercrystalline matrix of Bevelander and Nakahara (1969)] separates adjacent

tablets. Early amino acid analyses showed that the matrix around the tablets

was enriched in Ala and Gly residues, a composition, which conferred to the

matrix hydrophobic properties, similar to that of worm silk. Additional ultra-

structural studies showed that nacre tablets were not homogeneous. In particu-

lar, Crenshaw and Ristedt (1975) evidenced that sulfated polysaccharides

were localized in the central part of nacre tablets. These organic compounds

were supposed to act as crystal nucleators. Mutvei (1979), by etching nacre

Figure 3 Structure of the two main molluscan nacre textures. (A and B) SEM pictures of the nacre of the freshwater bivalve U. pictorum.

(C and D) SEM pictures of the nacre of the gastropod Haliotis tuberculata (bar scales ¼ 10 mm). (E) The ‘‘brick‐wall’’ model of bivalvian

nacre. (F) The ‘‘columnar’’ model of gastropod nacre. These simplified models, adapted from Nakahara (1991), do not take in account the

existence of pores in the interlamellar organic matrix, the substructures of nacre tablets, and the existence of a thin ACC layer around

the tablets. The constituents are: E, the secreting mantle epithelium; S, the organic sheets; SS, the newly formed surface sheets; Cr, the

aragonite crystals; T, the top of the newly formed crystals.

224 Marin et al.

tablets with a glutaraldehyde‐acetic acid solution, observed extremely complex

structures such as twinning patterns or concentric growth lamellae.

One of the first ‘‘modern’’ nacre models was proposed by Bevelander and

Nakahara (1969). The ‘‘compartment model’’ supposed that the aragonite

nacre tablets grow in a preformed mold made from the interlamellar matrix.

The main drawback of this model was to ignore all the organic ingredients of

the matrix, namely chitin, hydrophobic ‘‘silk‐fibroin‐like’’ proteins and

above all, acidic Asp‐rich proteins. The next model, published in the early

1980s by Weiner and coworkers, integrated all the recent findings of that

time: an acidic template for nucleating crystals (Weiner and Hood, 1975),

acidic macromolecules for inhibiting the crystal growth (Wheeler et al.,

1981), specific amino acid sequences (Asp‐rich) for chelating calcium ions

(Weiner, 1979, 1983), sulfated polysaccharides for attracting calcium ions

(Addadi et al., 1987). In this model, the insoluble framework was constituted

by a chitin core taken in sandwich between two hydrophobic silk fibroin‐likesole, on top of which lay a �‐sheet of Asp‐rich soluble proteins. The soluble

polyanionic sheet was supposed to function as a template by nucleating

aragonite crystals, while covalently bound acidic polysaccharides were sup-

posed to concentrate calcium ions at the vicinity of the template. The growth

of the crystal was stopped by the addition of an inhibiting layer of acidic

macromolecules on top of the newly formed tablets. The successive steps of

nucleation and inhibition were explaining the regularity and repetitiveness of

nacre. Because the aragonite tablets nucleated and grew on an organic

template, this crystal growth model was assimilated to heteroepitaxy.

B. Recent Nacre Models and Evolving Views

The heteroepitactic model for nacre achieved a frank success for more than a

decade, until AFM and TEM observations of the columnar nacre of the

abalone showed that holes, of diameter comprised between 5 and 50 nm,

were present in the interlamellar matrix (SchaVer et al., 1997; Song et al.,

2003). This finding suggested that nacre platelet grows in continuity with the

underlying ones, through mineral bridges, and not by heteroepitaxy. Song

et al. (2003) calculated that each platelet exhibits about 1400–1900 holes.

Holes had been observed previously, in particular by Mutvei (1969) and

Nakahara (1991), in gastropod and bivalve nacre. At that time, it was

suggested that the interlamellar holes facilitate the passage of mineral pre-

cursors for filling the empty compartments. So far, we do not know whether

the presence of holes is a general feature of nacre or represents particular

cases. Furthermore, it is still unclear whether they are mineral bridges or just

holes of a sieve, for allowing diVusion of the organic and mineral precursors

to the site of mineralization.


Another drastic evolution of the topography of the model occurred about

6 years ago, when Levi‐Kalisman et al. (2001) observed the nacre of the

bivalve Atrina with cryo‐TEM in the hydrated state. The changes go as

follows: �‐chitin is the highly ordered polymer, which gives the framework

that organizes the orientation of the crystals; the silk fibroin‐like proteins,

which are highly insoluble in their final state, are secreted as a disordered gel;

minerals grow in this gel and push it aside when they laterally extend; the gel

comprises also clusters of acidic macromolecules that are involved in nucle-

ating crystals. This model was nicely reshaped and integrated in a dynamic

perspective (Addadi et al., 2006), which presents the formation of nacre in

four stages: (1) assembly of the matrix (chitin, then silk gel); (2) formation of

the first mineral, ACC; (3) nucleation of aragonitic tablets via polyanionic

polymers; and (4) growth of the tablets, first in thickness (until reaching the

top interlamella) then laterally.

Although there is a general consensus on the fact that nacre tablets grow

from their center and expand laterally until reaching the confluence with

neighboring tablets, the ultrastructure of single‐nacre tablets remains un-

clear. Histochemical observations of Nautilus nacre by Nudelman et al.

(2006) confirmed the old finding of Crenshaw and Ristedt (1976), that is,

the concentration of reactive groups (carboxylate), presumably involved in

nucleating aragonite, in the center of single tablets. In addition, a zonation

was observed, which consisted of, from the tablet center to the periphery, a

central ring‐shaped area rich in sulfates, an intermediate zone rich in carbox-

ylate, and finally a tablet‐surrounding matrix rich in carboxylates and sul-

fates. Another recent paper (Nassif et al., 2005) showed that the nacre tablets

of the abalone were coated by an extremely thin layer (3‐ to 5‐nm thick) of

ACC. This layer may be a stabilized remnant of the transient ACC phase,

described by Addadi et al. (2006). A possible scenario for a single‐tabletgrowth suggests a lateral tablet expansion and the subsequent expulsion of

the gel. The process is driven by hydrophobic interactions. By doing so, the

organic ‘‘impurities’’ progressively concentrate in a front at the interface

between the mineral and the gel. During this growth phase, the transient

ACC is replaced by aragonite. When the centrifugal front meets a similar

front of the neighboring tablet, the degree of impurities becomes so high that

ACC is stabilized, which prevents further crystallization of aragonite.

At higher magnification, single‐nacre tablets exhibit a remarkable hierar-

chical architecture, which is somehow diYcult to conciliate with what has

been described above. Nacre tablets have fractal properties in the sense

that they exhibit diVerent levels of substructures that reproduce the same

motif: these are, for example, flat nanobuilding blocks (Oaki and Imai, 2005),

or ‘‘nanotablets’’ of 30‐ to 180‐nm long and less than 100‐nm thick, that

self‐assemble and self‐orientate. The laminated structure of single‐nacretablets has also been observed independently by Rousseau et al. (2005).

226 Marin et al.

AFM studies by the same authors suggest that the nanograins that constitute

each platelet are encapsulated in a continuous network of an organic intra-

crystalline phase (Rousseau et al., 2005). This phase looks like a foam, which

suggests that the early steps of tablet formation are performed in an emul-

sion. Reticulate circular imprints of an organic framework at triple junctions

between mature platelets in bivalve nacre have also been observed (Rousseau

et al., 2005). They are supposed to localize the spot where the new tablets

grow.

Clearly, since the beginning of the twenty‐first century, the nacre model

knows a complete revolution and requires the integration of diVerent levelsof observation, from micrometric to nanometric scales. The future topo-

graphic models will have to consider the fine architecture of the matrix, the

sequence of the secretory events, as well as purely crystallographic and

geometrical considerations, such as crystal competition.

C. Prism Models

Beside the well‐studied nacre, prisms constitute another key model and an

important shell texture found most frequently, but not exclusively, in mollus-

can outer shell layer, in particular, in gastropods, cephalopods, and bivalves.

Like nacre, prisms are supposed to be an archaic type of shell texture. Because

prisms are often associated to nacre, it has been proposed that nacre evolved

through simple horizontal partitioning of vertical prisms (Carter and Clark,

1985; Taylor, 1973). This appealing idea, based on simple geometric consid-

erations, needs to be reevaluated with accurate crystallographic and bio-

chemical criteria. This may help to understand the transition from one

microstructure to the other and to reconstitute primitive shell textures. It is

interesting to notice that prism‐like or palisade‐like minerals, with growth

axis perpendicular to the growth plan, represent an extremely common and

fast strategy found by diVerent biomineralizing systems (brachiopods, mol-

lusks, eggshell) for filling a space with minerals. In a first approximation,

prismatic textures exhibit many similarities with purely chemical crystal

growth. However, as we briefly show here, this view is probably oversimpli-

fied, and the deposition of prisms, similarly to nacre ones, is finely regulated

over diVerent scales.Prisms are calcitic or aragonitic needles of various lengths and diameters,

from the thin oblique calcitic prisms of the edible mussel, M. edulis, to the

large‐sized calcitic prisms (‘‘simple’’ prism type), developed perpendicularly

to the shell surface, among the fan mussel Pinna nobilis, or the aragonite

prisms of the freshwater mussel, Unio pictorum. Prisms of the outer shell

layer are secreted on the inner surface of the periostracum, at the growing

shell edge. They grow inward by the accretion of crystal units. They are


enveloped by an organ ic insol uble and hydropho bic sheath, whi ch form s a

hone ycomb ‐ like fram ework . This sheath is not homogeneo us, but compo sed

of at least three layer s ( Dauphin, 2002; M arin et al ., 2007 ). It mainta ins all

the prism s toget her an d allows a certa in flexibi lity of the structure. When

isolated from their org anic envelopes , prism s compri se an intr acrystall ine

organic fraction. In calci tic prism s, this matrix is pa rticular ly acidi c ( Marin

et al ., 2005 ).

Like for nacre, the form ation of prism s is far from being eluci dated. Fr om

Grigo r’ev (1965) , it is well known that prism ‐ like cryst als can be obtaine d bypurely ch emical ‘‘com petition for sp ace’’ cryst al growth. The star ting point

includes nuc leation spots, more or less unifor mly spread on a surfa ce, from

which spherul ites grow concen trically. W hen the spherul ites come into con-

tact, their gro wth is co nstrained in one direction, perpendicul ar to the

surfa ce. This ha ppens in natural environm ents wi thout the ne ed of a sophis-

ticated organic templ ate. Comp etition for space can be easil y simu lated

( Ubukata , 1994, 1997 ). By certain aspec ts, the prism gro wth in moll usks

looks like crystal compet ition. In a simila r way, the early step of molluscan

prism co nstruction is the form ation of spherul ites in the inn er surfa ce of the

perios tracum . This has be en clear ly sho wn for the bival ves, Pinna nobilis

( Cuif et al ., 1983 ) and Lampro tula sp. (C heca and Rodri guez ‐ Navar ro, 2001).

How ever, a compe tition for space phe nomeno n might occu r only in the early

steps of prism form ation (disapp earance of minut e prisms just below the

perios tracum ; see Checa et al ., 2005 ), but may not de scribe accurat ely the

subsequ ent steps of prism grow th, mainly for two reasons : the she aths are

form ed before the prism miner al infilling and the growth is con strained by

the organic shea ths a round the prisms.

Anothe r aspect that renders a simp le ‘‘com petition for space’ ’ mod el

inapprop riate is the mult iscale struc ture of prism s and their strik ing com-

plexi ty. A wel l‐ known exampl e is that of the ‘‘s imple type’ ’ calciti c prism s of

Pinna nobil is. Optical ly, each prism of P. nobilis be haves like a mon ocrystal,

with a singl e extincti on when obs erved wi th polari zed ‐ analyze d light (Cui fet al ., 19 83 ). How ever, enzymat ic treatment of the prism preparat ion shows

that each prism is consti tuted of a pile of flat crysta l uni ts, which can be

entire ly dissoc iated after pyrolys is. These cryst allites are the grow th units .

They are separated from each other by an organic intracrystalline template.

Within a pile, they are perfectly positioned according to their a, b, and c axes.

In spite of looking homogeneous, these crystallites are composed of subdo-

mains, emphasized by enzymatic treatments (Cuif et al., 1983) or immunos-

taining (Marin, unpublished data). These subdomains might as well be

composed of nanocrystal aggregates. Similarly to P. nobilis, ultrastructural

observation of the prisms of Cristaris plicate (Tong et al., 2002) revealed a

complex lacelike framework of intracrystalline matrix. Checa et al. (2005)

hypothesized that the interprismatic ‘‘honeycomb‐like’’ sheaths are formed

228 Marin et al.

by interfacial tensions that occur in a precursor liquid–liquid emulsion. This

elegant hypothesis needs further testing.

Is there a unique prism model? Nothing is less certain. We describe similar

objects by using a single terminology. However, many ultrastructural studies

show that significant diVerences occur, even in closely related taxa, as shown

in Fig. 4. The best example is that of Pinna nobilis and Pinctada margaritifera

(Cuif et al., 1991; Dauphin, 2003). In cross sections (perpendicular to the

growth axis), the prisms of Pinna nobilis look homogeneous and behave like

monocrystals. On the contrary, those of Pinctada margaritifera exhibit sinu-

ous intraprismatic membranes (particularly well visible by SEM, after etch-

ing) that separate the section in domains, and these domains do not have the

same crystallographic orientation. Another case is the prismatic outer layer

of Unio, which is absolutely diVerent from the two types cited above: the

prisms of Unio are composed of single‐crystal fibers radiating from spher-

ulites (Checa and Rodriguez‐Navarro, 2001; Cuif et al., 1983). Clearly,

important eVorts need to be put in the elucidation of the prism growth and

to relate it to the biochemical properties of the associated matrix.

Figure 4 Prismatic microstructures among bivalve shells. (A) Unio pictorum. (B) Anodonta sp.

For A and B, the aragonitic prisms (above) are in contact with the nacreous layer. (C) Oblique

thin calcitic prisms of the edible mussel Mytilus edulis. (D) Calcitic prisms of Atrina rigida.


IV. Molluscan Shell Proteins: Characterization of TheirPrimary Structure

In parallel to the structural studies on the diVerent shell textures, a consider-

able eVort was realized, in the last three decades, for identifying the diVerentmacromolecules that constitute the shell matrix and for obtaining informa-

tion on their primary structures. In most of the cases, these macromolecules

were analyzed after the dissolution of the mineral phase. Until now, the most

commonly used reagents are EDTA, a calcium‐chelating agent, which is

eVective at neutral pH, weak dilute acids, acetic or formic acids, or rarely

dilute hydrochloric acid. Our preference goes to cold dilute acetic acid,

progressively added to the cleaned shell powder suspended in Milli‐Qwater, until reaching pH 4, the decalcification process being performed at

4 �C (Marin, 2003). We assume that this procedure minimizes protein degra-

dation and precludes the formation of macromolecular aggregates, as EDTA

does. Other extraction processes include a soft—but long—demineralization

of the powder on a cation‐exchange resin (Albeck et al., 1996), or extraction

with water (Pereira‐Mouries et al., 2002). However, in this latter case, the

most strongly mineral‐linked macromolecules are not extracted.

The decalcification procedure yields two organic fractions, one soluble in

the decalcifying solution, the other one strongly insoluble. The ratio between

the two fractions can considerably vary: while the soluble fraction represents

between 0.03 and 0.5 wt %, the insoluble fraction varies in greater propor-

tions: from 0.01% (in some crossed‐lamellar neogastropods for instance) to

4–5 wt % of the shell of the abalone or of the nautilus! Usually, the second

fraction is discarded by centrifugation. In some cases, the insoluble fraction

may be partially dissolved by using strong denaturing agent (urea) and/or

by heating. The soluble extract can be cleared from decalcification salts by

ultrafiltration or dialysis. It can be further fractionated according to standard

biochemical techniques, electrophoresis, or chromatography (gel perme-

ation, ion exchange, aYnity). However, because molluscan shell proteins

have a nonstandard behavior due to polydispersity, multiple anionic charges,

posttranslational modifications, classical fractionations usually fail in resolv-

ing the soluble matrix in discrete macromolecules. This technical obstacle,

found also with several other calcified tissues, pestered for more than

two decades the life of researchers involved in biomineralization studies!

This explains in particular why the first partial amino acid sequence from a

mollusk shell was obtained in the early 1990s (Rusenko et al., 1991), and the

first full‐length sequence only in 1996 (Miyamoto et al., 1996).

The search for the primary structure of molluscan shell proteins benefited

from the major technical advances in molecular biology, in particular, from

the possibility to use degenerate oligonucleotide probes deduced from short

230 Marin et al.

partial N‐terminal or internal amino acid sequences. This allowed fishing out

the corresponding transcript by RT‐PCR or by cDNA library oligoscreen-

ing. Another strategy successfully developed by us was to use polyclonal

antibodies raised against shell matrix for screening expression cDNA

libraries (Marin et al., 2003a). On the 43 fully known sequences, 39 were

obtained via molecular biology and only 4 by direct protein sequencing. It is

predictable that the number of shell proteins will ‘‘explode’’ in the near

future, owing to the increasing number of fully sequenced genomes

(Livingston et al., 2006), and in the absence of genomic data, owing to EST

technique applied on shell ‘‘secretome’’ (Jackson et al., 2006).

In this chapter, we give a brief description of the diVerent shell proteins,one after the other. Complementary information can be retrieved in Marin

and Luquet (2004), in Matsushiro andMiyashita (2004), and in the review of

Zhang and Zhang (2006). What was possible few years ago, when the number

of identified and named proteins was still reasonable, would be fastidious and

redundant. We deliberately choose to present the known molluscan shell

proteins in three groups, according to their theoretical isoelectric point

(Fig. 5). The first group comprises proteins, the pI of which is below 4.5,

18

160

140

80

60

40

20

00 2 4 6 8 10 12

�20

108

93

1

24

5

67

1314

16

1512

11

1719 22

21MW

pI

Figure 5 Graphical representation of the distribution of the molecular weights (MW) of all

known molluscan shell proteins versus their isoelectric point (pI ). The theoretical MW and pI

were computed (http://www.expasy.ch/tools/pi_tool.html) after identification and removal of the

signal peptide (http://www.cbs.dtu.dk/services/SignalP/).□¼ proteins associated with aragonite;

◆ ¼ proteins associated with calcite; (� ¼ protein associated with both aragonite and calcite

(1 ¼ aspein; 2 ¼ Asp‐rich proteins; 3 ¼ MSP‐1; 4 ¼ MSP‐2; 5 ¼ MSI31; 6 ¼ prismalin‐14;7 ¼ N‐14/N16/pearlin/perline proteins masking AP7 and AP24; 8 ¼ MSI60; 9 ¼ mucoperlin;

10 ¼ nacrein from P. fucata; 11 ¼ MSI7; 12 ¼ dermatopontin; 13 ¼ tyrosinase‐like1;14 ¼ nacrein from T. marmoratus; 15 ¼ perlucin; 16 ¼ shematrin proteins; 17 ¼ perlustrin,

18 ¼ lustrin A; 19 ¼ perlwapin; 20 ¼ N‐66; 21 ¼ tyrosine‐like2; 22 ¼ KRMPs).

http://www.expasy.ch/tools/pi_tool.html

http://www.cbs.dtu.dk/services/SignalP/


the second group comprises proteins with a pI in the range 4.5–7, and the

third group comprises proteins with a pI above 7. We are fully aware that this

grouping is artificial and arbitrary but practical in the absence of clearly

identified protein families.

A. Extremely Acidic Shell Proteins

This first group, the most homogeneous one, comprises the most acidic shell

proteins (Marin and Luquet, 2007). The existence of such proteins is known

since the pioneering work of Weiner and Hood (1975), followed by extremely

precise chromatographic characterization (Weiner, 1979, 1983). However,

for the reasons described above, they were the most diYcult to purify. In

particular, they do not stain correctly on SDS‐PAGE (Marin et al., 2001).

Because of their negative charge, they are even suspected to diVuse readily

out of the electrophoresis gel, and they subsequently require a double fixation

(Gotliv et al., 2003). The two first full‐length sequences of very acidic proteins

deduced from their transcript were published in 1997 (MSI31; Sudo et al.,

1997) and in 2001 (MSP‐1; Sarashina and Endo, 2001). Today, this group

comprises only six proteins (Table I).

One striking feature of these proteins is their association with calcitic bio-

minerals rather than aragonite.MSI31, aspein, prismalin 14, andAsp‐richwereretrieved from calcitic prism textures, and MSP‐1 and MSP‐2 (SP‐S) fromfoliated calcite. The finding that acidic proteins are preferentially associated

with calcite in mollusk shell is not new: Hare (1963) already noticed that ‘‘the

organicmatrices from the calcite layers have a consistently higher ratio of acidic

tobasic aminoacids than the aragonitic shell units.’’ The reasonof this selection

is intriguing, but remains unknown.

Another feature associated with very acidic molluscan shell proteins is that

they are all singularly enriched in Asp residues. The ‘‘choice’’ for this amino

acid, rather than Glu, is remarkable, although poorly understood. It may

meet stereochemical requirements, the Asp side chain being shorter than that

of Glu. Because of their high amount of Asp residues and the side chains are

negatively charged under physiological conditions, these proteins are sup-

posed to easily bind calcium ions. They consequently belong to the group of

low‐aYnity, high‐capacity calcium‐binding proteins (Maurer et al., 1996),

which implies that they do not exhibit the typical ‘‘high‐aYnity, low‐capacity’’canonical calcium‐binding domains, such as EF‐hand (Kretsinger, 1976).

Their moderate aYnity for calcium is compatible with a reversible binding

of calcium ions.

MSI31 is a Gly‐rich protein of the insoluble matrix, and supposed to be

primarily a ‘‘framework’’ protein (Sudo et al., 1997), because of the 10 short

poly‐Gly blocks, distributed mainly in the N‐terminal domain. It exhibits

Table I Unusually Acidic Molluscan Shell Proteins (with a Theoretical Isoelectric Point Below 4.5)a

Protein Name Species

Microstructure

(polymorph) MW (kDa) pI (%AspþGlu)

Swiss‐Prot/TrEMBL

Accession

Number References

BIVALVIA Aspein Pinctada

fucata

Prisms (calcite) 39.3/41.2 1.67 (61.9) Q76K52 Tsukamoto et al.,

2004

MSI31 Pinctada

fucata

Prisms (calcite) 32.85/31 3.8 (14) O02401 Sudo et al., 1997

Prismalin‐14 Pinctada

fucata

Prisms (calcite) 11.9/13.5 4.24 (10.5) Q6F4C6 Suzuki et al., 2004

MSP‐1 Patinopecten

yessoensis

Foliated

(calcite)

74.6/76.4 3.34 (22.8) Q95YF6 Sarashina and

Endo, 1998, 2001

MSP‐2/SP‐S Patinopecten

yessoensis

Foliated

(calcite)

27.9/29.8 3.48 (22.3) Q6BC34 Hasegawa and

Uchiyama, 2005

Asp‐rich protein 1 Atrina rigida Prisms (calcite) 6.6/8.5 3.34 (50.8) Q5Y821 Gotliv et al., 2005

Asp‐rich protein 2 Atrina rigida Prisms (calcite) 15/17 2.89 (52.8) Q5Y822 Gotliv et al., 2005

Asp‐rich protein 3 Atrina rigida Prisms (calcite) 16.5/18.4 2.75 (60) Q5Y823 Gotliv et al., 2005

Asp‐rich protein 4 Atrina rigida Prisms (calcite) 18/19.9 2.73 (56.2) Q5Y824 Gotliv et al., 2005


Asp‐rich protein 6 Atrina rigida Prisms (calcite) 18.2/20 2.72 (59.2) Q5Y826 Gotliv et al., 2005



Asp‐rich protein 9 Atrina rigida Prisms (calcite) 18.2/20 2.72 (59.2) Q5Y829 Gotliv et al., 2005

Asp‐rich protein 10 Atrina rigida Prisms (calcite) 20/21.8 2.68 (60) Q5Y830 Gotliv et al., 2005

aThe sequences of all these proteins were deduced from their respective transcript sequence. Prismalin‐14 was also biochemically characterized. In the

MW column, the first number corresponds to the molecular weight of the protein without its signal peptide, and the second one to the unprocessed protein.


acidic C‐terminal motifs (6 XSEEDY, where X is D or E, and Y is M or T).

The acidic domainmay be involved in nucleating crystals, or binding calcium,

but this has not been tested. The hydrophobic N‐terminus may adopt a

�‐sheet conformation.

MSP‐1, the second unusually acidic protein of the Japanese scallop Pati-

nopecten yessoensis, is enriched in Ser, Asp, and Gly residues and exhibits a

modular structure, with a short‐basic domain, close to the N‐terminus and

two GS domains that alternate with D‐rich domains (Sarashina and Endo,

1998, 2001). The two Asp‐rich domains fit with the initial model of Weiner

and coworkers since they exhibit DGS and DS motifs. They also present

numerous DD repeats. All these motifs are suspected to bind calcium ions

or to interact with calcite crystals. In addition, several serine residues are

putatively phosphorylated or glycosylated. MSP‐1 exhibits homologies with

dentin phosphophoryns, very acidic proteins of the teeth. Recently was

found MSP‐2, also called SP‐S, another shell protein of the Japanese scallop

(Hasegawa and Uchiyama, 2005). MSP‐2 is a Ser‐Gly‐Asp‐rich protein of

the scallop, which exhibits 91% identity with MSP‐1. It may represent a

shortened variant of MSP‐1.The third unusually acidic protein is aspein, a protein of the pearl oyster

Pinctada fucata. The composition of aspein is remarkable since Asp residues

represent 60.4% of the whole protein, and its theoretical pI is 1.67, which would

make it the most acidic protein found to date! The two other abundant amino

acids are Gly (16%) and Ser (13%). The main body of aspein is composed of

58 poly‐Asp blocks (of 2–10 Asp residues long) interspersed by SG dipeptides.

Some Ser residues may be phosphorylated. Aspein exhibits some simila-

rities with aspolin, phosphophoryn, and bone sialoprotein‐binding protein.

The primary structure of aspein suggests that it is a high‐capacity, low‐aYnity

calcium‐binding protein.Asp‐rich is a family of 10 related proteins, composed of the following

domains, from N‐ to C‐terminus: hydrophobic (signal peptide), short basic,

acidic 1, variable acidic, DEAD repeats, and acidic 2. Interestingly, the acidic 1

domain has a high homology with calsequestrins, calcium‐binding proteins

from cardiac and skeletal muscles, and may consequently bind calcium. The

variable acidic domain exhibits long stretches of poly‐Asp. TheDEADmotif is

also found in helicases, enzymes that separate the two DNA strands. Asp‐richand aspein are closely related proteins since they share 48% homology.

The last member is prismalin‐14, the single one characterized both at

protein and at transcript levels (Suzuki et al., 2004). Prismalin was extracted

from the insoluble hydrophobic framework of the prismatic layer of Pinctada

fucata. It is a G/Y‐rich protein, representing, respectively, 27.6% and 20% of

the amino acid composition. It exhibits PIYR repeats, a G/Y‐rich region and

N‐ and C‐terminal D‐rich calcium‐binding regions. It inhibits the precipita-

tion of calcium carbonate in vitro and induces morphological changes of

234 Marin et al.

calcite crystals. Prismalin 14 as well as aspein and MSI31 are specifically

expressed in the mantle, in the region, which secretes the prism matrix

(Takeuchi and Endo, 2005). In addition, the expression levels of these three

proteins are correlated, which suggests that they are secreted at the same time.

B. Moderately Acidic Shell Proteins

As shown in Table II, the second group corresponds to acidic proteins, with

pIs between 4.5 and 7. In this disparate group, one finds gastropod and

bivalve nacreins, MSI60, MSI7, the N14/pearlin/N16 family, mucoperlin,

AP7, AP24, a tyrosinase‐like protein, perline, and a snail dermatopontin.

First of this list is nacrein, by many aspects the most‐studied molluscan

shell protein family (Matsushiro andMiyashita, 2004; Miyamoto et al., 1996,

2003, 2005; Miyashita et al., 2002; Takeuchi and Endo, 2005). Nacrein, the

first protein whose primary structure was deciphered, was initially found as a

50‐kDa EDTA‐soluble protein of the nacreous layer of the Japanese pearl

oyster Pinctada fucata. Later on, a similar protein (nacrein), with an identical

N‐terminus, was found in association with the prismatic layer (Miyashita

et al., 2002). In situ hybridization studies (Miyamoto et al., 2005; Takeuchi

and Endo, 2005) shows that nacrein is ubiquitous and displays probably the

same functions in the two layers. Nacrein is also the first protein, which was

proved to work as an enzyme. Nacrein exhibits several GXN repeats (where

X is frequently D, N, or E), flanked by two carbonic anhydrase‐like sub-

domains. Strikingly, these two subdomains have a relatively high homology

with human carbonic anhydrase II (CA). In addition, the first nacrein CA‐like domain exhibits the three histidine residues involved in zinc binding,

typical of CA. A full‐length recombinant nacrein inhibits the in vitro precipi-

tation of calcium carbonate. Interestingly, the recombinant nacrein, which

lacks the central GXN repeat domain, does not exhibit this property, where-

as the repeat domain, tested alone, has a strong inhibiting ability (Miyamoto

et al., 2005). Another nacrein was found in the shell of the gastropod Turbo

marmoratus (Miyamoto et al., 2003). This moderately acidic protein has a

longer repeat domain, constituted of GNmotifs. At last, N66, retrieved from

the Australian pearl oyster Pinctada maxima, belongs also to the family

(Kono et al., 2000). It exhibits two carbonic anhydrase subdomains, and a

longer central repeat domain constituted by 46 GXN motifs interspersed by

12 GN motifs. The repeat domain of N66 is much less acidic than the one of

nacrein, which implies that N66 has a basic pI.

Among the acidic shell proteins, MSI60 is an insoluble framework protein

retrieved initially from the nacreous layer. It exhibits 11 poly‐Ala blocks and

39 poly‐Gly blocks dispersed throughout the sequence. The poly‐Ala blocks

confer to MSI60 some homologies with spider silk fibroins. The MSI60

Table II Moderately Acidic Molluscan Shell Proteins (with a Theoretical Isoelectric Point 4.5 � pI � 7)a


Microstructure

(Polymorph) MW (kDa)

p I (% Asp

þ Glu)

Swiss ‐Prot/TrEMBL

Accession

Number References

BIVALVIA N14 Pinctada maxima Nacre (aragonite) 13.7/16.4 4.8 (15.8) Q9NL39 Kono et al., 2000

Nacrein Pinctada fucata Nacre (aragonite) 48.2/50.1 6.85 Q27908 Miyamoto et al., 1996

N16/Pearlin Pinctada fucata Nacre (aragonite) 12.8/15.4 5.14

(16.9)

O97048 Samata et al., 1999;

Miyashita et al.,

2000

MSI60 Pinctada fucata Nacre (aragonite) 61.7/60 4.8 (5.9) O02402 Sudo et al., 1997

MSI7 Pinctada fucata Prisms (calcite) 7.3/9.3 5.98 (2.6) Q7YWA5 Zhang et al., 2003a

Tyrosinase‐ likeprotein 1

Pinctada fucata Prisms (calcite) 56.3/58.3 6.5 (9.3) A1IHF0 Nagai et al., 2007

Perline Pinctada

margaritifera

Nacre (aragonite) 13.6/16.2 4.7 (15.8) Q14WA6 Montagnani

et al., 2006

Mucoperlin Pinna nobilis Nacre (aragonite) 65.4/66.7 4.87 (9.5) Q9BKM3 Marin et al., 2000

AP7 Haliotis rufescens Nacre (aragonite) 7.6/9.9 5.43

(12.1)

Q9BP37 Michenfelder et al.,

2003

AP24 Haliotis rufescens Nacre (aragonite) 17/19.6 5.53

(13.6)

Q9BP38 Michenfelder et al.,

2003

GASTROP Nacrein Turbo

marmoratus

Nacre (aragonite) 56/57.6 5.76

(10.7)

Q8N0R6 Miyamoto et al., 2003

Dermatopontin Biomphalaria

glabrata

Crossed‐lamellar

(aragonite)

16.6

(no s.p.)

6.33

(10.8)

P83553 Marxen et al., 2003b

aThe sequence of dermatopontin was obtained by direct protein sequencing. All the other proteins were retrieved from their transcript sequences. In the

MWcolumn, the first number corresponds to themolecular weight of the protein without its signal peptide, and the second one, to the unprocessed protein.

s.p. ¼ signal peptide.

236 Marin et al.

N‐terminus contains two Asp‐rich domains and four Cys residues, and the

C‐terminus contains one short Asp‐rich domain and one Cys residue. In situ

hybridization shows that MSI60 is specifically associated with the secretion

of nacre (Takeuchi and Endo, 2005). In a paper (Asakura et al., 2006), the

first Asp‐rich domain (16 residues among which 11 are acidic) of MSI60 was

introduced between diVerent Ala/Gly‐rich domains derived from silk

fibroins, and the conformation of the resulting peptides, studied by NMR

spectroscopy. It was shown that the calcium‐binding ability of the acidic

domain was the most eVective when the flanking domains had a �‐sheetconformation.

Another protein family is represented by a series of low molecular and

moderately acidic proteins of the nacre of the pearl oyster. This protein

family is studied by two independent Japanese groups, a reason that explains

why this family is called either N14/N16 (Kim et al., 2004; Kono et al., 2000;

Samata et al., 1999, 2003) or pearlin (Miyashita et al., 2000, 2003;

Matsushiro and Miyashita, 2004; Matsushiro et al., 2003a,b) All the mem-

bers of this family diVer by few amino acids. They are enriched in Gly, Tyr,

and Asn residues and exhibit Gly‐Asn repeat sequences in addition to four

short acidic domains (3–12 residues). They exhibit a putative phosphoryla-

tion site, in addition to a heparin‐binding domain. Pearlin, as defined by

Miyashita, is a calcium‐binding protein, but this ability is conveyed

by a covalently bound sulfated polysaccharide (Miyashita et al., 2003).

Samata et al. (1999) observed that N16 in solution inhibits the in vitro crystal

growth but induces the formation of aragonite when fixed on the water‐insoluble matrix. By using a diVerent experimental device, Matsushiro et al.

(2003a,b) observed that pearlin was able to make protein complex with pearl

keratin. Only in the presence of CaCO3 saturated solution containing Mg2þ,the complex induced the formation of aragonite. The dissociated complex

lost this ability, while the reconstituted complex recovered this property,

suggesting that the polymorph selection is determined at supramolecular

level. The N16/pearlin proteins are specific of nacre matrix, as it was shown

by diVerent techniques, Northern blot (Kono et al., 2000; Samata et al., 1999)

and in situ hybridization (Takeuchi and Endo, 2005). In addition, quantita-

tive RT‐PCR showed that the levels of expression of N16 and nacrein are

correlated (Takeuchi and Endo, 2005). Recently, perline was obtained from

the Polynesian pearl oyster Pinctada margaritifera (Montagnani et al.,

submitted for publication). Perline has 93% homology with N14.

Mucoperlin, a protein retrieved by immunoscreening of the bivalve Pinna

nobilis cDNA library (Marin et al., 2000, 2003a), belongs to a completely

diVerent protein family. This protein is composed of three domains: a short

N‐terminus, a central region made of 13 tandem repeats of 31 amino acid

residues each, and a C‐terminal part enriched in serine. The presence of

central tandem repeats, the presence of Pro and Ser residues in the repeat


domain, the numerous putative O‐glycosylation sites, and the demonstration

that mucoperlin is glycosylated are criteria that aYliate mucoperlin to the

mucin family. Mucins are ubiquitous proteins associated with epithelial

tissues. They exhibit several functions in connection with their ability to

form gels: they are involved in epithelial lubrication, act as eYcient barriers

against chemical aggressions, but they also play a role in cell signaling.

Mucoperlin was the first protein that was directly localized in the shell,

owing to a polyclonal antibody raised against a recombinant mucoperlin.

Mucoperlin is only present in the nacreous layer. Classical immunohistolo-

gical staining (Marin et al., 2000) as well as immunogold technique (Marin,

unpublished data) showed that the protein is concentrated around the nacre-

ous tablets, in particular on the lateral sides rather than on the top/bottom

layers. Mucoperlin may be one of the constituents of the gel‐like matrix

described by Addadi et al. (2006), which is pushed aside when nacre tablet

grows laterally.

Two other protein s, named AP 7 and AP24, wer e isol ated from the ED TA ‐soluble matrix of the nacreous layer of the abalone Haliotis rufescens

( Michenf elder et al ., 2003 , 2004). They are solub le and mo derately acidi c

(pI around 5.4–5.5). They aVect the growth of calcite crystals in vitro. The

calcium carbonate mineral interaction domain of AP7 and AP24 is localiz-

ed in the first 30 amino acid residues of their N‐termini, as deduced

from conformation studies by NMR spectroscopy and CD spectrometry

(Kim et al., 2004; Wustman et al., 2004). AP7 and AP24 are considered to

act as crystal‐modulating proteins.

Another protein, MSI7, is a moderately acidic small protein (pI 5.98) of

the Japanese oyster Pincta da fucata (Zhan g et al ., 2003a) . Its N ‐ terminus is

highly homologous to the N‐terminal domain of the much more acidic

MSI31. In particular, MSI7 harbors the Gly‐rich sequence, which may be

involved in calcium binding. The expression of the MSI7 transcript suggests

that this protein is involved in the formation of the nacreous and prismatic

layers. MSI7 aVects the in vitro growth of calcite crystals. In the same

organism was recently retrieved a tyrosinase called Pfty1, from the outer

prismatic layer (Nagai et al., 2007). Tyrosinase, a particular phenoloxydase,

is a copper‐containing enzyme that binds oxygen. It is involved in the oxida-

tion of phenol groups of tyrosine residues, which results in the formation of

melanin. Pfty1 exhibits a conserved copper‐binding site, which suggests that

its oxidative function is active. Pfty1 seems to be involved in the pigmenta-

tion of the prismatic layer and may be included in the prismatic layer. It is

possible that Pfty1 plays a role in the defense of the pearl oyster against

parasites.

Another protein was directly purified and sequenced from the HCl‐solubleshell matrix of the freshwater snail B. glabrata, a model that has also been

studied for developmental purposes (see above, Section II). Interestingly, the

238 Marin et al.

protein sequence represents the first one found in association with the crossed‐lamellar structure of a modern gastropod (Marxen and Becker, 1997; Marxen

et al., 2003b). Called dermatopontin, this protein has a striking homology with

vertebrates and invertebrate dermatopontins, a family of extracellular matrix

proteins involved in binding decorins and TGF‐� . It was suggested that thesnail dermatopontin has a role in organizing spatially the shell matrix. Whether

this protein has also a role in cell signaling is unknown. Another interesting

feature about dermatopontin is the presence of a unique N‐linked saccharide inthe first third of the sequence. The structure of this pentasaccharide has been

determined, but its exact function is unknown.

C. Basic Shell Pro teins

Cont rarily to the most acidic protei ns, protei ns wi th a basic pI were rather

unexpected as components of the molluscan shell matrix (Table III). However,

their existence could have been predicted by comparison with the sea urchin

spicule model, for which several basic proteins were discovered one decade ago

(Killian and Wilt, 1996; Wilt et al., 2003).

The first found basic protein is also the most complex in it its primary

structure and the most ‘‘popular,’’ that is, the most often cited as a ‘‘model’’

protein for biomineralization. This is lustrin A (She n et al., 1997), an insoluble

protein retrieved from the nacreous layer of the abalone H. rufescens. From all

the molluscan shell proteins, lustrin A is the single protein, which established a

clear link between a primary structure and the overall mechanical property of

nacre. Lustrin A has been indeed the subject of diVerent structure–functionstudies (B.L. Smith et al., 1999; Wustman et al., 2002, 2003a,b; Zhang et al.,

2002). The sequence of lustrin A comprises, from its N‐ to its C‐terminus, nine

Cys‐rich modules (79–89 amino acid residues) interspersed by eight Pro‐richmodules (19–30 amino acid residues), followed by a long GS domain, a short

D‐rich domain, a Cys‐rich modules, a short basic domain, and a protease

inhibitor‐like C‐terminus. Interestingly, the first Pro‐rich domain exhibits

homology (53%) with a collagen I‐� chain fragment. AFM pulling studies

have shown that the interlamellar matrix, which is supposed to contain lustrin

A, exhibits a typical sawtooth force‐extension curve with hysteretic recovery(B.L. Smith et al., 1999; Zhang et al., 2002). This mechanical behavior is

explained by the successive stretching of springs (the Cys‐rich modules), sepa-

rated by spacers (the Pro‐rich modules), when an increasing stretching force is

applied to the molecule. In addition to the elastomeric properties of lustrin A,

the GS domain (GS loop) provides further a highly flexible domain, while the

basic C‐terminal domain (RKSY) may interact with other macromolecules,

and the short acidic one (D4) may be a mineral‐b i nd i n g r eg io n ( Wu st ma n

et al., 2003a). Clearly, lustrin A is a multifunctional protein.

Table III Basic Molluscan Shell Proteins (with a Theoretical Isoelectric Point > 7)a

Protein

Name Species

Microstructure

(Polymorph) MW (kDa) pI

Swiss‐Prot/TrEMBL

Accession

Number References

BIVALVIA N66 Pinctada maxima Nacre (aragonite) þPrisms (calcite)

59.8/62.4 8.66 Q9NL38 Kono et al., 2000

Shematrin‐1 Pinctada fucata Prisms (calcite) 30.3/31.9 9.04 Q1MW96 Yano et al., 2006







KRMP 1 Pinctada fucata Prisms (calcite) 9.5/11.5 9.6 Q1AGW0 Zhang et al., 2006c

KRMP 2 Pinctada fucata Prisms (calcite) 9.8/11.8 9.4 Q1AGV9 Zhang et al., 2006c

KRMP 3 Pinctada fucata Prisms (calcite) 9.8/11.8 9.4 Q1AGV8 Zhang et al., 2006c

Tyrosinase‐like prot. 2

Pinctada fucata Prisms (calcite) 54.4/56.5 9. A1IHF1 Nagai et al., 2007

GASTROP Lustrin A Haliotis rufescens Nacre (aragonite) 140/142.2 8.13 O44341 Shen et al., 1997

Perlustrin Haliotis laevigata Nacre (aragonite) 9.3 (no s. p.)b 8.02 P82595 Weiss et al., 2001

Perlucin Haliotis laevigata Nacre (aragonite) 18.2 (no s. p.) 7.15 P82596 Mann et al., 2000

Perlwapin Haliotis laevigata Nacre (aragonite) 14.5 (no s. p.) 8.62 P84811 Treccani et al., 2006

Perlinhibin Haliotis laevigata Nacre (aragonite) 4.79 (no s. p.) 8.26 P85035 Mann et al., 2007

aThe sequences of perlustrin, perlucin, perlwapin, and perlinhibin were obtained by direct protein sequencing. The other proteins were retrieved from

their transcript sequences. In the MW column, the first number corresponds to the molecular weight of the protein without its signal peptide, and the

second one to the unprocessed protein.bs.p. ¼ signal peptide.

240 Marin et al.

Two other basic protei ns were charact erized , one from the Austral ian

pearl oyster, Pincta da marg aritif era , the other one from the Ja panese one,

Pincta da fucata . The first one is N66 ( Kono et al ., 2000 ), whi ch belongs to the

nacrei n fami ly. N66 is describ ed in Se ction IV.D. The second one is Pfty2, the

second tyros inase ‐ like protei n ( Nagai et al., 2007 ). Pfty2 e xhibits high ho-

mology with Pf ty1 (54% in 493 resi dues overla p). The hom ology is maximal

in the cen tral region. Simi larly to Pfty1, Pfty2 con tains a conserved co pper ‐binding sit e. Its presum ed functi on is the oxidat ion of Tyr resi dues, for

pigme nting the shell (melanog enesis).

Four proteins were purified from the nacreous layer of the abalone, Haliotis

laevigata, and directly sequenced. These proteins are perlustrin (Weiss et al.,

2000, 2001), perlucin (Mann et al., 2000; Weiss et al., 2000), perlwapin

(Treccani et al., 2006), and perlinhibin (Mann et al., 2007). Perlustrin is a

small protein, the sequence of which exhibits similarities to vertebrate insulin‐like growth factor‐binding protein (IGF‐BP) sequences (40% homology), in

particular a pattern of 12 Cys residues, spread along the sequence. The IGF‐BPs represent a family of proteins, which bind growth factors of the insulin

type. In vitro tests showed that perlustrin binds IGFs with a good aYnity and

insulin with a low aYnity. Perlucin is an N‐glycosylated protein (via oneasparagine residue), the sequence of which exhibits similarities with calcium‐dependent lectins (C‐type). Perlucin has some sequence similarities with

asialoglycoprotein receptors. Functional tests showed that the carbohydrate

recognition domain (CRD) of perlucin has a broad specificity, but binds

particularly mannose and galactose. In vitro studies showed that perlucin

promotes the nucleation of CaCO3 crystals. Further AFM investigations

(Blank et al., 2003) confirmed that perlucin is able to accelerate the

nucleation of CaCO3 layers on top of calcite surfaces. This protein is

incorporated as an intracrystalline component of the neosynthesized crystals.

The third abalone shell protein, perlwapin, contains 3 repeats of 40 amino acid

residues, which are very similar to the whey acidic proteins (WAP), a family of

proteins characterized by a conserved pattern of 8 characteristically spaced Cys

residues. These residues are involved in disulfide bond formation. Perlwapin

exhibits also a high homology with the C‐terminus of lustrin A. Perlwapin has a

polymorphism in its sequence (three variants). AFM studies on calcite surfaces

indicated that it is a potent inhibitor of calcium carbonate precipitation by

binding selectively to distinct step edges, thus preventing the crystal layer from

growing further. It is suggested that perlwapin inhibits the growth of certain

crystallographic planes. At last, the fourth protein perlinhibin inhibits the

growth of calcite and induces the formation of aragonite (Mann et al., 2007).

The functional relationships between perlucin, perlustrin, and perlwapin,

perlinhibin and the insoluble matrix are unclear: it was recently shown that

this latter is able to induce alone growth of flat oriented tablets that mimic nacre

(Heinemann et al., 2006). Another protein, perlbikunin, has been characterized

from the nacre layer of the abalone, but no sequence data are available yet.


Recently, a set of diVerent proteins was identified in a cDNA library

constructed from mantle tissues of the Japanese pearl oyster Pinctada fucata.

With one exception, all these newly found proteins have a pI above 9, con-

stituting thus the most basic proteins found to date, in association with the

molluscan shell. They are distributed in two families: K‐rich matrix proteins

(KRMPs) and shematrin. KRMPs represents a group of three small proteins

with amolecular weight of 10 kDa (Zhang et al., 2006c). These three proteins,

of 98–101 residue long, diVer only by few amino acids. They are Lys‐Gly‐Tyrrich. Apart the signal peptide, their primary structure is composed of a Lys‐rich domain (40 amino acid residues long), which comprises also all the Cys

and Trp residues, and a C‐terminus (39 amino acid long) enriched in Gly and

Tyr, which comprises also a short acidic motif. The Gly‐Tyr‐rich region

exhibits some homologies with few quinone‐tanned proteins, which suggests

that the Tyr residues may be oxidized in DOPA in the mature protein. The

Lys‐rich domain may interact with negatively charged ions (bicarbonate) or

acidic matrix proteins. The protein is expressed only in the mantle edge,

corresponding to the secretion of the prisms. The structure of KRMPs sug-

gests that their function is to link the acidic soluble proteins to the hydropho-

bic framework of the prisms. The second family of basic proteins, shematrin,

comprises seven members, of molecular weights between 25 and 33 kDa

(Yan o et al. , 2 006). With one excepti on (shematr in ‐ 5, pI ¼ 7.7) , all these

proteins have a pI between 9 and 10.3. They all exhibit Gly‐rich domains,

constituted of short motifs of the typeXGnX (with 2� n� 6 andX¼L/Y/A/

V/I/M). The C‐terminus of all shematrins ends with an RKKKY, RRKKY,

or RRRKYmotif. Surprisingly, theGly‐rich domain of shematrin‐2 is almost

identical to that of the acidicMSI31 (98% homology in a 227 residue overlap),

but their respective C‐terminus diVers completely. On the other hand, the

beginning of the C‐terminal half of shematrins exhibits a high homology

(above 60% on 26 residues) with the C‐terminal Gly‐rich region of KRMPs.

Shematrin‐5 is the single protein of that family to contain an acidic domain,

which has homology with aspein. All shematrin transcripts are expressed in

the mantle edge of P. fucata, which indicates that this protein family is

expressed as components of the prism matrix. Protein sequencing of a urea

extract of the water‐insoluble prism matrix showed that shematrins belong to

this fraction. It is suggested that shematrins play a role as framework proteins.

D. Partially Characterized Shell Proteins

Beside fully sequenced proteins, an increasing number of shell proteins have

been partially characterized, as shown in Table IV. These proteins, or protein

fractions, enter four categories: the first category comprises proteins that have

been partially sequenced and well characterized, in particular on gel or by

Table IV Partially Characterized Shell Proteins

Protein Name Species Microstructure Features

Swiss‐ProtAaccession

Number References

A. Partial

Sequence(s)

BIVALVIA Nacrein‐like protein Pinctada fucata Nacre (aragonite) 415 aa A0ZSF2 Norizuki, M.

(submission author)

WSM peptides Pinctada

margaritifera

Nacre (aragonite) Repeats of Q/

KGGGI/L

or Q/

KGAGI/L

Bedouet et al., 2006

p20 Pinctada

maxima

Nacre (aragonite) 21 (N‐terminal

sequence)

Bedouet et al., 2001

Nacrein‐like protein Pinctada

maxima

Nacre (aragonite) 421 aa A0ZSF3 Norizuki, M.

(submission author)

Nacrein‐like proteinP1

Patinopecten

yessoensis


(submission author)

Nacrein‐like proteinP2

Patinopecten

yessoensis


(submission author)

Nacrein‐like proteinC1

Crassostrea

nippona


(submission author)

Nacrein‐like proteinC2

Crassostrea

nippona


(submission author)

RP‐1 fraction Crassostrea

virginica

Foliated (calcite) 59 aa

(6 internal

fragments)

Donachy et al., 1992;

Rusenko et al., 1991

RP‐1 fraction Adamussium

colbecki

Foliated (calcite) 34 aa

(6 internal

fragments)

Halloran and

Donachy, 1995

Caspartin Pinna nobilis Prisms (calcite) 62 aa; 17 kDa

(SP);

putative

poly‐D‐domain

Marin et al., 2005, 2007

Calprismin Pinna nobilis Prisms (calcite) 61 aa; 38 kDa

(SP)

P83631 Marin et al., 2005

P12, P16 Mytilus

californianus

Prisms (calcite) D‐P‐T‐Drepeats in

the two

proteins

Weiner, 1983

45‐, 21‐, and 5‐kDa

proteins

Mytilus edulis Prisms (calcite) þnacre

(aragonite)

N‐terminal

sequences:

13 aa

(45 kDa); 14

aa (5 kDa)

Keith et al., 1993

N‐terminal

sequences:

30 aa

(21 kDa)

Q9TWS3 Keith et al., 1993

55‐, 20‐, and15‐kDa proteins

Atrina vexillum Nacre (aragonite) N‐terminal

sequences:

13 aa

(55 kDa); 15

aa (20 kDa);

6 aa

(15 kDa)

Zhao et al., 2003

60‐, 32‐, and12‐kDa proteins

Nautilus

pompilius

Nacre (aragonite) N‐terminal

sequences:

13 aa

(60 kDa); 10

aa (32 kDa);

19 aa

(12 kDa)

Zhao et al., 2003

Dermatopontin2 Satsuma

japonica

Crossed‐lamellar

(aragonite)

51 aaa Q50K83 Sarashina et al., 2006

Dermatopontin2 Mandarina

aureola

Crossed‐lamellar

(aragonite)


(Continued)

Table IV Continued

Protein Name Species Microstructure Features

Swiss‐ProtAaccession

Number References

GASTROPODA Dermatopontin1 Mandarina

aureola

Crossed‐lamellar

(aragonite)


Dermatopontin2 Euhadra

peliomphala

Crossed‐lamellar

(aragonite)


Dermatopontin1 Euhadra amaliae Crossed‐lamellar

(aragonite)



herklotsi

Crossed‐lamellar

(aragonite)



herklotsi

Crossed‐lamellar

(aragonite)


Dermatopontin1 Euhadra brandtii Crossed‐lamellar

(aragonite)


Dermatopontin2 Euhadra brandtii Crossed‐lamellar

(aragonite)


Dermatopontin2 Biomphalaria

glabrata

Crossed‐lamellar

(aragonite)


Dermatopontin1 Biomphalaria

glabrata

Crossed‐lamellar

(aragonite)


Dermatopontin3 Lymnea

stagnalis

Crossed‐lamellar

(aragonite)



stagnalis

Crossed‐lamellar

(aragonite)



stagnalis

Crossed‐lamellar

(aragonite)


ACLS40 Strombus

decorus

persicus

Crossed‐lamellar

þ prisms

(aragonite)

40 kDa (SP);

25 aa

(2 internal

fragments)

Pokroy et al ., 2006b

B. No sequence BIVALVIA 30‐kDa protein Atrina rigida Nacre (aragonite) Aragonite‐nucleating

protein

Gotliv et al., 2003

P10 Pinctada fucata Nacre (aragonite) 10 kDa (SP) Zhang et al., 2006a

Periostracin Mytilus edulis Periostracum 20 kDa (SP);

55% Gly

Waite et al., 1979

GASTROP AP8 Haliotis

rufescens

Nacre (aragonite) AP8‐�(8.7 kDa;

SP); AP8‐�(7.8 kDa;

SP)

Fu et al., 2005

Dermatopontin Biomphalaria

glabrata

Crossed‐lamellar

(aragonite)

61.2 kDa (SP);

11 aa

Marxen and Becker,

1997

aPartial sequences obtained by RT‐PCR.

For most of them partial sequences are available. We also indicate nonsequenced proteins (bottom), which have been only biochemically characterized.

SP ¼ SDS‐PAGE, in this case, the indicated molecular weight is evaluated from the electrophoretic migration of the protein.

246 Marin et al.

chromatography. This is the case of RP‐1, p20, caspartin and calprismin, P12

and P16, and ACLS40. The second category includes proteins that are known

only by a partial sequence. This is the case of the proteins of Nautilus

pompilius, Atrina vexillum, M. edulis, and the 61‐kDa protein of B. glabrata.

The third category includes putative proteins retrieved from partial nucleo-

tide sequences, such as diVerent unpublished nacrein‐like proteins and homo-

logues of dermatopontins in diVerent gastropods. The last category comprises

biochemically characterized proteins, for which no sequence is available yet.

In the first category, one finds RP‐1, EDTA‐soluble phosphoproteins

extracted from foliated calcitic shells, the American oyster C. virginica

(Donachy et al., 1992; Rusenko et al., 1991), and the Antarctic scallop

Adamussium colbecki (Halloran and Donachy, 1995). These Asp‐rich pro-

teins inhibit the in vitro precipitation of calcite, but this eVect is conveyed by

phosphorylated Ser residues, and not by Asp. They exhibit similarities with

phosphophoryns. RP‐1 proteins are related to very acidic proteins (MSP‐1,MSP‐2, aspein, Asp‐rich). Similarly, two Asp‐rich proteins, which were

purified from the calcitic layer of the American mussel,Mytilus californianus,

would also enter this protein family (Weiner, 1983). Two proteins were

retrieved from the calcitic prisms of the fan mussel Pinna nobilis (Marin

et al., 2005, 2007). One, caspartin, is a 17‐kDa Asp‐rich unglycosylated

protein. It binds calcium ions with a low aYnity, is a strong inhibitor of

calcite precipitation in vitro, and dramatically aVects the shapes of calcite

crystals in interference tests. Caspartin is abundant in the prism‐solublematrix, but is also present in the nacre‐soluble matrix, but in much lesser

amount (about eight times less). Caspartin polymerizes and may form high

molecular weight complexes. One polyclonal antibody raised against purified

caspartin showed that this protein is localized within and around the prisms

(intracrystalline and intercrystalline). Calcite crystals grown in the presence

of caspart in exhibi t a sli ght modificat ion of their lattice pa rameters (Pok roy

et al ., 2006a), the highest variation being recorded for the c axis. A secon d

prism‐soluble protein, calprismin, was also characterized from Pinna nobilis.

Calprismin is an acidic glycoprotein of 38 kDa, for which one‐fifth of the

sequence is known. It is enriched in Ala (16%), Asx (15%), Thr (12%), and

Pro (12%). Although the N‐terminus is characterized by a particular 4 Cys

pattern, it does not exhibit clear homology. The characterization of its

glycosyl moieties is in progress. P20 is a protein extracted from the nacre of

Pinctada maxima (Bedouet et al., 2001). Its N‐terminus is enriched in Tyr

residues. P20 can form oligomers constituted by six monomers linked togeth-

er by disulfide bridges. Recently, a 40‐kDa protein was extracted from the

aragonitic crossed‐lamellar shell of the gastropod Strombus decorus persicus

(Pok roy et al ., 2006b). Aragon ite cro ssed‐ lame llar structure pro tein 40

(named ACLS40) is Glu/Asp/Ala rich (13.5%, 12.3%, and 12%, respectively).

One of its internal sequences has some homology with a vertebrate


dimet hylaniline mon ooxygenase, but the signi ficance of this similarit y is

obscu re. Interesti ngly, ACLS 40 ha s the ability to stabili ze the thermo dynam-

ically unstabl e CaCO3 polymor ph vaterite . At last, severa l short pep tides of

molec ular weight inferior to 1 kDa were extra cted from the nacre of Pincta da

marg aritifera (Be douet et al., 2006 ). Some of these peptide s are rich in Gly

resid ues. Whether these pe ptides have a c ell ‐ signaling fun ction or whet herthey are de gradation products of shell protei ns is not known.

The second category comprises proteins, which are only known by partial

N‐terminal or internal sequences. One 61‐kDa protein, retrieved from the

crossed‐lamellar freshwater gastropod B. glabrata (Marxen and Becker,

1997), does not have clear relationship. A similar situation is encountered

with three proteins of the edible mussel, M. edulis of 45, 21, and 5 kDa,

respectively (Keith et al., 1993), and of uncertain aYnities. More recently,

short sequences of three proteins (60, 32, and 12 kDa, respectively) extracted

from the cephalopod,N.pompilius (Zhao et al., 2003),were obtained, but donot

share significant homology with other shell proteins. One of the three proteins

(55, 20, and 15 kDa), extracted from the nacro‐prismatic bivalve, A. vexillum

(Zhao et al., 2003), has homology with the enzyme phosphodiesterase, but this

similarity may be fortuitous.

The third category includes newly found proteins, the sequences of which

were deduced from their incomplete transcript sequences. Diverse nacrein‐likeproteins have been retrieved from the Japanese edible oyster, Crassostrea

nippona, and the scallop, P. yessoensis (Norizuki, unpublished data). These

fresh data confirm that nacrein is a true protein family, the members of which

possess highly conserved domains. A second group of shell proteins was

retrieved from diverse gastropods, by amplifying cDNAs with degenerate

oligonucleotide probes encoding dermatopontin (Sarashina et al., 2006).

Incomplete sequences of 13 new dermatopontins were obtained, which exhibit

short conservedmotifs. The analysis of the expression pattern of the transcripts

showed that some dermatopontins are ubiquitous, whereas others are only

expressed in the mantle tissue. Only these second ones may be real shell

proteins, but this needs to be demonstrated at protein level. At last, the EST

work of Jackson et al. (2006) on the abalone, Haliotis asinina, produced a

huge amount of sequences encoding secreted proteins. Among these, few are

suspected to encode shell proteins.

The last category of partially characterized shell proteins corresponds to

proteins, which have been completely purified, usually by chromatography

or electrophoresis, and for which no sequence data are available. This implies

that these proteins have been characterized at amino acid composition

level. Several analyses performed on shell matrix ‘‘fractions’’ (HPLC frac-

tions ), which con sist of mixtu res of di Verent protein s ( Albeck et al ., 1993 ;

Almeida et al., 2000; Per eira ‐ Mo urie s et al., 2002; Sa mata, 1990; Wheeler

et al., 1988), are excluded from this category. The first purified protein is

248 Marin et al.

periostracin, a soluble precursor of the periostracal layer of the edible mussel,

M. edulis (Waite et al., 1979). Periostracin is a 20‐kDa basic and hydrophobic

protein with a unique amino acid composition consisting of 55% Gly, 10%

Tyr, and 2.2%DOPA. Periostracin is a ‘‘transient’’ protein, which is oxidized

and crossed‐linked to form the insoluble periostracum as soon as it is secreted

by the periostracal groove. Since the very complete work of Waite et al., no

more characterization was performed on that protein. Cariolou and Morse

(1988) described two proteins, one 43‐ and one 54‐kDa polypeptides,

obtained in native conditions from juvenile and adult nacre tissues of the

abalone H. rufescens. Both were enriched in Asx and Gly residues, but the

first was more acidic and the second was more hydrophobic. Similar amino

acid compositions were obtained from the same species for proteins purified

in denaturing conditions (Belcher and Gooch, 2000). Two proteins of 16 and

20 kDa were obtained. They were predominantly constituted of Gly and Asx

residues. Fu et al. (2005) characterized one small acidic protein, called AP8,

from the nacre of the same abalone species. AP8 has two variants of 8.7

(AP8‐�) and 7.8 kDa (AP8‐�). Both are enriched in Asx (35%) and Gly (40%)

residues. Interestingly, they represent the first aspartate‐rich proteins found

in association with aragonite. AP8 proteins modify drastically the shape of

calcite crystals grown on Kevlar. In the nacre‐soluble matrix of the abalone,

they may be the most eVective crystal‐shaped modifiers since the soluble

matrix depleted of AP8 has a minor eVect on calcite crystals. Other acidic

proteins were extracted from the nacreous layer of the bivalve Atrina rigida

(Gotliv et al., 2003). When tested in vitro together with chitin and silk (Falini

et al., 1996), they induce the formation of ACC prior to its transformation

into aragonite. At last, a small protein extracted from the nacre of Pinctada

fucata was characterized (Zhang et al., 2006a). This hydrophobic protein,

named p10, is Gly rich (37%) and contains high amounts of Leu (16%) and

Ala (13%) residues. In in vitro assay, p10 accelerates the precipitation of

calcium carbonate and induces the formation of aragonite needlelike crys-

tals. In addition, p10 when tested on two cell lines (MRC‐5 and MC3T3)

increases the ALP activity, suggesting that p10 may play a key role in

triggering cell diVerentiation for producing bone tissues.

E. Other Molluscan Proteins: The Extrapallial Fluid and the Mantle

This long list of proteins, fully or partially characterized, would not be

complete without considering other proteinaceous constituents that may

be, directly or indirectly, involved in the formation of the shell (Table V).

These proteins are located in two diVerent compartments (Fig. 2): the

extrapallial space and the mantle epithelium.

Table V Mantle Epithelium and Extrapallial Fluid (EP) Proteinsc


Localization/

Expression Features

Swiss‐ ProtAccession

Number References

Ferritin Pinctada fucata Mantle 23.6 kDa (cDNA); involved in iron

incorporation into the shell

Q7YW43 Zhang et al., 2003b

Gs� Pinctada fucata Mantle and gill 44.3 kDa; G protein (�‐subunit s class) Q6TP31 Chen et al., 2004

Calmodulin Pinctada fucata Mantle and gill 16.8 kDa (cDNA); 4 EF‐hands; involvedin Ca transport and secretion

Q6EEV2 Li et al., 2004

CaLP Pinctada fucata Mantle 18.4 kDa (cDNA); 4 EF‐hands Q3BD18 Li et al., 2005

OT47/Tyrosinase Pinctada fucata Mantle EC 1.14.18.1; 47 kDa (cDNA); involved

in periostracum formation

Q287T6 Zang et al., 2006b

CA/Carbonic

anhydrase

Pinctada fucata Mantle EC 4.2.1.1; 38 kDa (SP) Yu et al., 2006

PSKH1 Pinctada fucata Mantle and gill Ca2þ/calmodulin‐dependent proteinkinase; 47 kDa (cDNA); involved in

calcium metabolism during nacre/pearl

formation

Q4KTY1 Dai et al., 2005

PFMG1a Pinctada fucata Mantle 15.7 kDa (cDNA); 2 EF‐hands; involvedin signal transduction during nacre/

pearl formation

Q3YL59 Liu et al., 2007

Calconectin Pinctada margaritifera Mantle 10.4 kDa (cDNA); 2 EF‐hands(1 complete, 1 partial domain)

Q1KZ60 Duplat et al., 2006

ESTs Haliotis asinina Mantle 530 sequences (mantle cDNA library); 25

clones present similarities with putative

biomineralization genes from the

L. scutum genome

DW986183

to

DW986511b

Jackson et al., 2006

Has‐Lustrin Haliotis asinina Mantle Fragment of 68 aa (1 of the 25 clones) A0S725 Jackson et al., 2006

EP Mytilus edulis Extrapallial

fluid

236 aa (sequence of the unprocessed

protein, from the cDNA)

P83148 Hattan et al., 2001;

Yin et al., 2005

aNine other PFMG genes have been sequenced (PFMG2 to PFMG12), the function of which does not appear related to shell formation or is unknown.

bGenBank accession numbers (all the sequences are not yet available in the Swiss‐Prot/TrEMBL databases).cThese proteins are putatively involved in shell formation, or in calcium metabolism in relation with the shell calcification. SP ¼ SDS‐PAGE, in this

case, the indicated molecular weight is evaluated from the electrophoretic migration of the protein.

250 Marin et al.

As recal led in Secti on II.B , the extra pallial space, local ized be tween the

outer mantl e epithelium and the grow ing shell, contai ns the extrap allial flui d

wher e the precurs ors of the shell minerali zation are sup posed to concentra te

and self ‐ assem ble in a precis e manne r. Thi s fluid ha s been ch emically char-

acter ized in a number of cases (Misogi anes and Chas teen, 1979; Moura et al.,

2000 ). How ever, cu riously, there are ha rdly any data on the pro tein co nsti-

tuents of this fluid . The single well ‐ch aracterize d extra pallial fluid pro tein isEP of the ed ible mussel, M . edulis . Thi s protei n was fir st identified as a major

compo nent of the fluid (56% of the total fluid pr oteins), purified an d bio-

chemi cally an alyzed (Hatt an et al., 2001 ), be fore be ing charact erized by

molec ular biology techn iques ( Yin et al., 2005 ). EP is a smal l acidi c glyco-

protei n (p I 4.4; M W 14 kDa with the glycos yl moieties , 12 kDa when degly-

cosyla ted) of 213 resid ues, whi ch is enriched in His (14% ), Asx (12% ), an d

Glx (13% ) resi dues. In nativ e con ditions, EP forms 28 ‐ kDa dimer s owing to

two Cys resi dues. EP is glycosylat ed via one asparag ine residue in the

N ‐ terminus. EP does not exhibit sequ ence homo logy with any known pro-

tein. EP possess es numerou s short acidi c motifs, suggest ing that it may

inter act with calci um ions. The fact that histidine is the most abund ant

resi due in the EP sequ ence is puzzli ng be cause none of the multiple amino

acid compositions of shell matrix detected this residue as a main amino acid.

On the other side, an earlier work (Hofmann et al., 1989) identi fied a

histidine‐rich calcium‐binding protein of the sarcoplasmic reticulum of the

rabbit muscle with a similar amino acid composition (His 13%; Asp 12%;

Glu 19%), suggesting that the positively charged His residue may also play a

functional (repulsive) role in the binding of calcium ions. Attempts to detect

EP in the shell were not successful so far. More generally, this finding points

out a limitation of searching only shell proteins. This implies that several

proteins, which are secreted in the extrapallial fluid for controlling the shell

formation process, may stay or degrade in the fluid, without being

incorporated as shell matrix components. If these ‘‘silent’’ and ‘‘transient’’

proteins exist, then they will be retrieved only by overlapping EST work on

secretome, as performed by Jackson et al. (2006), and shell matrix proteomics.

Beside extrapallial fluid proteins, several proteins may play a very impor-

tant role in the process of shell formation. For example, these are proteins

specific of the molluscan mantle. The analysis of mantle proteins is far

beyond the scope of this chapter and would require a review by itself.

However, because we suspect mantle‐specific proteins to be key players in

the physiology of calcification, we mention some of them in Table V. As

illustrated, most of them have been very recently characterized. Many are not

secreted because they do not exhibit signal peptides. They may play impor-

tant intracellular functions, such as the regulation of intracellular calcium. In

this group, one finds calconectin, a calcium‐binding protein of the mantle of

the Polynesian pearl oyster (Duplat et al., 2006), calmodulin (Li et al., 2004),


or ferr itin (Zhang et al ., 2003b ). Concer ning ferr itin, it is interest ing to note

that this protein may be involved in iron concentration in the mantle cells

and its subsequent incorporation in the shell. A tyrosinase (Zhang et al.,

2006b), putatively involved in the formation of the periostracum, has also

been retrieved. We can predict that the number of mantle‐specific proteins

will dramatically increase soon, as illustrated by the impressive EST work of

Jackson et al. (2006b) on juvenile mantle tissue of the abalone, Haliotis

asinina. This work generated 530 sequences, encoding nonsecreted and

secreted proteins, from which 85 encode secreted proteins. Clearly, the

coming years will require accurate in silico analyses.

F. Remarks on Molluscan Shell Proteins

Since 1996, when the first full sequence of nacrein was published, the number

of identified proteins has grown exponentially, and we do not see any reason

why this evolution would cease. However, the expanding corpus of published

shell protein sequences does not hide some obscure gaps in our knowledge of

these proteins. At first, a number of shell proteins have been characterized

at the transcriptional level but have not been clearly identified as constituents

of the shell matrix. Nacrein, N14/N16, prismalin‐14, dermatopontin, muco-

perlin, AP7, and AP24 represent the few cases where the link between the

transcript and the protein was established. Moreover, we dispose now of

more than 40 protein sequences, but we still have a rather unprecise idea of

their respective putative functions, which are mainly deduced from primary

structure analysis and homology search with known proteins. In several cases,

because of low homologies (for example, the N‐terminus of calprismin), this

approach is ineVective. In addition, many of these proteins exhibit sites for

posttranslational modifications, but we have a poor idea of the biochemical

characteristics of these modifications, which can so drastically aVect the

properties of the protein core. Dermatopontin is so far the single example

where its sugar moieties were precisely characterized. The discrepancy

between the topographic models, presented in Section III.B, and the known

proteins is another problem, and a validation of the model by direct localiza-

tion of each shell matrix protein on and within the shell biominerals would be

extremely helpful. The last important drawback is that our knowledge is

almost entirely limited to ‘‘economically interesting models,’’ in particular,

the pearl oyster and the abalone, twomollusks, which exhibit nacro‐prismatic

shell textures. It is very unlikely that these two studied species are representa-

tive of the huge phylogenetic and textural diversity of the phylum Mollusca,

which comprises more than one hundred thousand living species.

In spite of these limitations, some general characteristics can be sketched.

As shown in Fig. 5, the molluscan shell proteins present indeed some

252 Marin et al.

parti cularities in their distribut ion accordi ng to their theoret ical molec ular

weigh t and their pI . Along the pI axis, most of the proteins associ ated with

calci te a re either very acidic (below 4.5) or basic, the single ex ception s being

MS I7 and Pfty1. On the c ontrary, most of the proteins associated wi th

aragoni te occu py a cen tral posit ion, in a p I range 4.5–7. 5, the exceptio ns

being perlus trin, perlwapin, and lustrin A. Along the y axis, the protein

dist ribution is bimodal , with a major ity of ‘‘smal l’’ protein s (34) in the

molec ular wei ght range 6.5–40 kDa, an d 9 pro teins above 48 kDa. Fr om

Fig. 5 an d sequen ce analys is, the outline of di Verent protei n famili es can be

rough ly sketche d. One dist inguish es the N14/ N16/p earlin family, which

form s a v ery hom ogeneou s grou p, the na crein fami ly, the shemat rins, the

KRM Ps. In the acidic group, the Asp‐ rich protei ns form a g roup by them-

selve s, which also includ e aspein as well. M SP‐ 1 and MSP ‐2 woul d c onstitutea dist inct group.

One charact eristic found in many of these proteins is their modular orga-

nization, each module corresponding to a functional domain. Consequently,

many of these proteins are multifunctional, a common feature found in

severa l protei ns of the extra cellular matr ix (Engel, 1991, 1996). Som e

domains are clearly identified, like the carbonic anhydrase‐like domains of

nacrein, the IGF‐BP domain of perlustrin, or the C‐type lectin domain of

perlucin. Many domains are composed of tandemly arranged repeats (muco-

perlin, MSI31) or of an alternance of two (Pro and Cys modules of lustrin A)

or three (SG, D, and K modules of MSP‐1) repeats. The repeats can be

extremely short: one residue (poly‐Gly, poly‐Ala, poly‐Asp, or poly‐Ser); tworesidues, like GS (lustrin A) or GN (nacrein); six residues (GGYGXX in

shematrin‐4/5; GGGGVI in shematrin‐3; XSEEDY in MSI31); several resi-

dues, like in mucoperlin (31 residues per repeat) in lustrin A (up to 88

residues in the Cys‐rich repeats), or in MSP‐1 (95 residues in the D modules).

These low complexity sequences can constitute an important part of the

protein sequence. Consequently, they considerably influence the overall

amino acid composition, which is dominated by few amino acids: Gly,

Asp, Ser, then Asn, Tyr, Ala, Pro, Cys, and Leu. The other consequence is

that these low‐complexity domains cannot be exploited for sequence com-

parison, and the fact that short segments of them match does not infer a

phylogenetic proximity. Inside families, sequence comparisons can produce

high similarities. But sequence comparison of proteins, from family to family,

does not produce significant similarities and the homologies on the full‐length sequences are generally low. Interestingly, we noticed that similar

short motifs, which are not necessarily of low complexity, can be found in

diVerent proteins (Marin et al., 2007), suggesting that these functional motifs

are reused as ‘‘building blocks’’ by diVerent mineralizing ‘‘mosaic’’ proteins

with diVerent functions. This implies that these proteins have been con-

structed by a genetic tinkering, which would have allowed the recombination

of short nucleotide sequences, for producing novel proteins.


A remarkable illustration of this genetic tinkering mechanism is given by the

shematrin‐2/MSI31 examples. The long structural hydrophobicN‐termini (227

residues) of these two proteins have 98% homology, but their C‐termini are

completely diVerent: that of shematrin is strongly basic and is supposed towork

as an anchoring domain, while that ofMSI31 is extremely acidic and would be

involved in nucleating crystals. If the diVerence in the C‐terminal sequence is

not due to a sequencing mistake, that is, a frameshift in the sequence reading,

then this homology is intriguing and calls for diVerent possible mechanisms by

which these proteins are constructed. Some explanations lie at the genome

organization level, the other at the transcripts level. Among the first one, exon

shuZing is a very likely mechanism (Gilbert, 1978), for creating new functions

from old ones. Exon shuZing is very common in the genes encoding extracellu-

lar matrix proteins (Kolkman and Stemmer, 2001; Patthy, 1996, 1999, 2003).

Exon shuZing is likelywhen the borders of an exon coincidewith the beginning

and end of the protein functional domain. The duplication, permutation, and

rearrangement of these exons result in novel genes with new functions. Exon

shuZing can occur through retrotransposition (Eickbush, 1999) or illegitimate

recombination (van Rijk and Bloemendal, 2003). In the present case, this could

be tested by looking in the genome ofPinctada fucatawhether the 227matching

N‐terminal residues of shematrin/MSI31 correspond to an exon. Another way

of explaining the partial homology of shematrin‐2 and MSI31 is the insertion/

deletion of one base in the duplicated gene, which leads to a frameshift during

transcription, and the synthesis of two proteins with diVerent C‐termini. At the

transcriptional level, alternative splicing can be inferred as another mode for

synthesizing two variants from a single gene, if the two exons that encode the

two C‐termini are placed in tandem in the gene. Alternative splicing of the

mRNA may then produce either shematrin‐2 or MSI31. This hypothesis can

also be tested, both at the genome and at the transcript levels.

There are still simple unanswered questions: how many proteins are

required for building a shell? This questionmay appear naive, but the answer is

not simple and has a lot to do with the technique used. In a monodimensional

gel, a soluble gel extract is usually characterized by the presence of few major

bands, in addition to several minor ones, embedded in a smear of nondiscrete

macromolecules (Marin et al., 2001). By this technique, about 10–15 proteins

can be visualized, in particular when the sensitive silver staining is used or

when accurate fixation method is employed (Gotliv et al., 2003). The same

extract tested on a bidimensional gel brings another answer. Some protein

bands, which appeared homogeneous in one‐dimension, can be constituted

of very diVerent proteins (Marie et al., 2007). Furthermore, to make the

problem a little bit more complicated, matrix proteins can exhibit several

posttranslational modifications, that is, phosphorylations, glycosylations,

sulfations. DiVerent phosphorylation patterns of a single protein will be

translated on the two‐dimensional gel by series of horizontally aligned

254 Marin et al.

spots of di Verent pI . A prelimina ry an swer can be given by assum ing that the

sea urchin spicul e and the moll uscan she ll syst ems exhibi t the same degree of

complex ity in their protei naceou s comp osition. If so, we can expect that 2D

gels will visualize few tens of diV erent protein spots in the solubl e shell matrix

( Killian an d W ilt, 1996 ). Wo rking at secretome level, by analyzing tran-

scri pts that encod e secret ed pro teins, brings a thir d mo re complet e answ er

( Jackson et al ., 20 06 ). Such studies reveal in parti cular the ‘‘transient an d

silen t’’ pro teins, whi ch are not incorp orated in the sh ell (some extra pallial

fluid pr oteins), as well as highly crossed ‐linked shell protei ns, which can notbe solubilize d an d analyze d by class ical bioch emical methods . At last, analy-

sis of the trans criptome will reveal severa l more protein s of the intra cellular

machi nery, involv ed in particu lar in the cell ular tra Yck ing, in the pro per

foldi ng of extra cellular matr ix componen ts.

V. Origin and Evolution of Mollusc an Shell Proteins

A. The Cambrian Origin of Mollu sk Shell Mine ralization

Puzzling questions concern the origin of these shell proteins. Where do they

come from? How were they recruited? Are they heavily constrained from an

evolutionary point of view? These questions have to be replaced in the general

context of the so‐called ‘‘Cambrian explosion.’’ Indeed, like most of the meta-

zoan lineages, mollusks started to mineralize at the dawn of the Cambrian

times, in a very short time interval, about 544 million years ago (Bengtson,

1992; Conway Morris, 2001). In the fossil record, the appearance of biologically

controlledminerals amongmetazoans, including silica, calciumphosphate, and

calcium carbonate skeletons, was themost visible aspect of the so‐called ‘‘Cam-

brian explosion,’’ by far the most important event in the metazoan world

(ConwayMorris, 1998; Knoll and Carroll, 1999; Shubin and Marshall, 2000).

As shown in Fig. 6, the mollusk fossil record indicates that the main classes

had representatives in the Cambrian (Lecointre and Le Guyader, 2001;

Runnegar, 1996), including polyplacophores (Matthevia, Upper Cambrian),

monoplacophores (Latouchella, Anabarella, Lower Cambrian), gastropods

(Kobayashiella, Upper Cambrian), cephalopods (Plectronoceras, Upper Cam-

brian), bivalves (Pojetaia, Fordilla, Lower Cambrian). Furthermore, it seems

that mollusks were able to exploit rapidly most of the design possibilities for

building their skeleton [see the ‘‘Skeleton Space’’ theory of Thomas et al.

(2000)]. Although there are no certainties on the Precambrian evolutionary

history of mollusks, the fossil record suggests that the phylum was also one of

the components of the well‐known Ediacaran fauna. The species Kimberella

quadrata is generally recognized as a shell‐less mollusk (Fedonkin and

Waggoner, 1997). Other Precambrian fossils, like the Chinese Circotheca

Vendian

Cambrian Ordovician

444 MY488 MY545 MY

Lower Middle Upper Lower Middle Upper

1

2

Solenogastra

Caudofoveata

Polyplacophora

Monoplacophora

Gastropoda

Cephalopoda

Bivalvia

Scaphopoda

Class

Shell-lessmolluscsof ediacara

Figure 6 Origin and phylogeny of the phylum Mollusca. The phylogeny of mollusks is that

proposed by Lecointre and Le Guyader (2001) in which Solenogastra and Caudofoveata occupy

a basal position. Node 1 represents the subphylum Eumollusca, whereas node 2 represents the

superclass Conchifera (shell‐bearing mollusks). Except scaphopods (first fossil record: Upper

Ordovician, 450 million years ago), all eumolluscs emerged in the Cambrian, bivalves and

monoplacophores emerging as early as the Lower Cambrian, about 540 million years ago,

and taking part of the ‘‘Cambrian explosion.’’


longiconica or the American Wyattia reedensis, are supposed to be mollusks,

although their taxonomic aYnities are still controversial.

Another argument which suggests that mollusks were already in existence

in the Precambrian comes from phylogenetic reconstructions based on mo-

lecular markers (18S rDNA). In general, all reconstructions agree with a

silent ‘‘revolution,’’ somewhere in the Proterozoic, the late part of the Pre-

cambrian. This revolution led to the identification of successive radiations

among the bilaterian metazoans: a first event isolating deuterostomians from

protostomians, a second event splitting proteostomians in lophotrochozoans

and ecdysozoans (Adoutte et al., 2000; Balavoine and Adoutte, 1998). What-

ever the radiation scenario is, this means that mollusks, like several other

metazoan phyla, acquired the capacity to form a mineralized exoskeleton far

after their emergence as a phylum. This has direct implications on the way the

‘‘molecular tool box’’ (including skeletal proteins) required for mineralizing

was recruited.

To explain how calcification was implemented in nonmineralized metazo-

ans, two ‘‘extreme’’ scenarios are possible: on one hand, calcification was

inherited from ancestral functions. If so, it resulted from the recruitment and

the orchestration of Precambrian functions, not related at all with minerali-

zatio n (Mari n et al. , 2003b). The best term inology for describing this process

is ‘‘exaptation,’’ a word invented byGould andVrba (1982), 25 years ago. The

alternative scenario implies that calcification was acquired independently by

256 Marin et al.

the diVerent metazoan lineages. If so, similarities are the result of adaptive

convergence. Such convergence would be explained by similar physical ‘‘con-

straints’’ that would ultimately drive the evolutionary process at the molecu-

lar level: secretion of an extracellular acidic template for crystal nucleation;

secretion of inhibitors for controlling crystal growth and allowing crystal to

grow only where needed. Let us examine these two hypotheses.

B. The ‘‘Ancient Heritage’’ Scenario

By many aspects, the ‘‘ancient heritage’’ scenario is ‘‘intellectually appeal-

ing,’’ although we admit that it is supported by a thin corpus of disparate

observations and laboratory data.

The first argument in favor of the ‘‘ancient heritage’’ hypothesis comes from

the fossil record. Detailed descriptions of shell textures of early mollusks (Feng

and Sun, 2003; Kouchinsky, 2000) indicate that, although far less numerous

than now, Cambrian shell textures were already complex and diversified. In

particular, it is striking to observe that Cambrian prismatic or nacreous tex-

tures resemble that of living species. This clearly suggests two remarks: first, the

number of textural combinations is limited and mollusks exploited rapidly all

the genetic possibilities given to them for building coherent assemblages of

crystals. Second, nacre and prisms, which are usually considered ‘‘primitive,’’

must be extremely ‘‘evolutionary’’ constrained. Nacre in particular is a strik-

ingly stable and perennial texture. Intuitively, the evolutionary constraint on

the texture may also apply on the macromolecules, which shape this texture. If

so, then nacre matrix is necessarily built from ‘‘ancient’’ proteins, and it is

tempting to establish a parallel between the molluscan nacre matrix and verte-

brate bone collagen, that is, an early invention of a successful molecular tool,

and its subsequent durability over the geologic ages.

In 1980, in a key paper, Lowenstam and Margulis assumed that the

appearance of calcium carbonate exoskeletons in the Lower Cambrian was

preceded by a phase during which the regulation of intracellular calcium ions

was set up. In other words, producing biominerals outside the cell requires

necessarily an accurate domestication of intracellular calcium fluxes. Calcium

acquired indeed a central position in the cell physiology. It is a second

messenger for diverse metabolic pathways; it is essential for muscle contrac-

tion, secretion, and cell adhesion. Intracellular calcium is stored in diVerenthighly structured cellular compartments (Pozzan et al., 1994), which are able

to quickly deliver calcium pulses when needed. The assumption of Low-

enstam and Margulis that the intracellular machinery required for handling

intracellular calcium was already finely tuned when animals started to calcify

suggests at least that parts of the intracellular calcifying machinery are

ancient.


Another point to mention concerns the serological comparisons estab-

lished in the last 20 years with antibodies elicited against diverse calcified

matrices. Remarkable immunologic interphylum cross‐reactivities were ob-

served in a number of cases: vertebrate to echinoderm (Veis et al., 1986),

echinoderm to prochordates (Lambert and Lambert, 1996), or mollusks to

brachiopods (Marin, unpublished data). Within the phylum Mollusca, we

also detected striking interclass cross‐reactivities (Marin et al., 1999), which

were related neither to taxonomy nor to shell microstructures. Although

cross‐reactivities may be fortuitous, due to similar overall topographies of

the epitopes, they may be also highly significant of the presence of short

conserved epitopes across calcifying phyla.

About 10 years ago, we hypothesized that epithelial mucus substances

might have been precursors of the calcifying matrices among mollusks and

corals (Marin et al., 1996). Our scenario called ‘‘anticalcification’’ was based

on the ‘‘functional duality’’ of acidic soluble matrices, that is, their ability to

inhibit crystal growth in solution, and to promote crystal nucleation, when

bound to a template. We assumed that, in the Proterozoic, epithelial mucus

substances were working as inhibitors of mineralization for precluding spon-

taneous calcium carbonate crystallization on ‘‘naked’’ ediacaran metazoans,

in a context of a heavily supersaturated ocean. At the Precambrian/Cambri-

an transition, the same mucus inhibitors could have been recruited to keep

crystallization in check. The fact that epithelial mucins (mucus proteins) are

extremely ubiquitous, that they are often associated with inhibiting systems

(salivary mucins, urinary mucins) or calcifying systems (gallbladder mucins),

and the finding of mucoperlin, a mucin‐like protein associated with the nacre

of the bivalve P. nobilis (Marin et al., 2000) emphasize the role and the

putative ancestry of this protein family in metazoan calcification.

Another striking example that further supports the idea of ‘‘ancient heri-

tage’’ is the bioactivity of nacre for bone repair (Atlan et al., 1997; Lopez

et al., 1992). In diVerent series of in vivo experiments, Lopez and coworkers

showed that nacre implants have the ability to promote bone repair without

provoking rejection. Their experiments were confirmed by Liao et al. (1997,

2000, 2002). Later on, Lopez and coworkers demonstrated that the water‐soluble nacre matrix was the fraction that contains the bioactive molecule(s)

(Almeida et al., 2001) and that this fraction induced the diVerentiation of

preosteoblastic cell lines (Rousseau et al., 2003). The most likely is that nacre

contains a diVusible signal transducing factor that can be recognized by

membrane receptors of bone‐forming cells, osteoblasts. So far, this factor

has not been identified, but putative candidates include bone morphoge-

netic proteins (BMPs), members of the TGF‐� superfamily. BMPs have

been identified in mollusks (Lelong et al., 2000, 2001; Matsushiro and

Miyashita, 2004), but it has not been yet demonstrated that these proteins

are incorporated within the shell matrix. Whatever the signaling molecule is,

258 Marin et al.

the fact that a molluscan signal transducer is able to activate a vertebrate

system pleads for the antiquity of the signaling function in biomineralization

(Westbroek and Marin, 1998).

Finally, the analysis of the primary structure of shell matrix proteins may

also contribute to suggest the antiquity of some conserved domains. The

most revealing examples are the carbonic anhydrase domains of nacrein and

N66. Carbonic anhydrases constitute a complex family of zinc‐containingenzymes, known in the three kingdoms, Archaea, Bacteria, and Eukarya

(K.S. Smith et al., 1999). They are subdivided in three classes designated � , � ,and � that evolved independently and do not exhibit sequence homologies.

All known metazoan carbonic anhydrases belong to the �‐class (Lindskog,1997), while those of the kingdom Bacteria belong to the �‐, �‐, and �‐classes.Archaea possess only�‐ and �‐classes of carbonic anhydrases (Smith andFerry,

2000). The high homology of nacrein with human carbonic anhydrase II

is puzzling. Because the reversible conversion of carbon dioxide into bicar-

bonate is an ancestral function, because this function is primordial in calcium

carbonate biomineralization (bicarbonate is one of the precursor mineral

ions for calcification), and because carbonic anhydrase domains have been

found in bivalve and gastropod nacres, it is hard to conceive that such a key

function in shell function results from a recent recruitment. Another point to

mention concerns the Asp‐rich domains. As mentioned by Tsukamoto et al.

(2004) and Gotliv et al. (2005), aspein as well as Asp‐rich proteins exhibit a

homology with calsequestrins, very acidic proteins of the sarcoplasmic retic-

ulum (Beard et al., 2004). Calsequestrins are high‐capacity, low‐aYnity

calcium‐binding proteins. They can bind up to 50 cations, in particular via

their short poly‐Dmotifs. Asp‐rich as well as aspein are supposed to display a

similar function. Few years ago (Marin et al., 2003b), we have suggested that

Asp‐rich domains of acidic molluscan shell proteins may derive from poly-

anionic domains of calsequestrins. Although our assertion is speculative, it

needs to be tested further. If it happened to be true, then it would give

consistency to the old intuition of Lowenstam and Margulis (1980) about

the prerequisite for calcification. All these combined data taken together

suggest that, at least, some shell components may be ancient and recruited

early in the evolution of the phylum.

C. The ‘‘Recent Heritage and Fast Evolution’’ Scenario

Facing the ‘‘ancient heritage’’ scenario, four recently published papers based

on unquestionable data demonstrate that, on the contrary, many metazoan

skeletal proteins evolved independently and that the calcification secretome

may have a much higher plasticity than expected.


One important paper from Livingston et al. (2006) has scanned the full

genome of the sea urchin Strongylocentrotus purpuratus, in search of all the

putative biomineralization proteins. Several sea urchin spicule proteins are

known from classical molecular biology approach (reviewed in Wilt et al.,

2003). From sequence homologies, and from the fact that biomineralization

protein genes are organized in clusters in the genome of S. purpuratus,

additional genes were retrieved. In total, the genes encoding spicule matrix

proteins constitute a small family of 16 sea urchin specific genes, which do not

have homology in other deuterostomes, that is, chordates and hemichordates.

Reversely, many of the vertebrate biomineralization proteins do not have

their equivalent in the sea urchin genome. This suggests that the mineralizing

matrices of echinoderms, on one side, and of vertebrates, on the other side, are

two ‘‘independent inventions.’’ Conversely, the protein components of the

twomatrices may have a unique origin, which has been completely obliterated

at the primary structure level, due to loose constraints and considerable

genetic drift.

In vertebrates, a rather similar conclusion was drawn by reviewing all the

members of the secretory calcium‐binding phosphoprotein family (SCPP)

(Kawasaki and Weiss, 2006). Members of this family, including three major

enamel matrix proteins, five proteins necessary for dentin and bone forma-

tion, milk casein, and salivary proteins, arose from a single ancestor by

tandem gene duplications (Kawasaki et al., 2004). Because of the huge

variability of the primary structures of the SCPP family members, it was

concluded that ‘‘while mineralized tissues are retained during vertebrate

evolution, the underlying genetic basis has extensively drifted.’’ This clearly

suggests that the real evolutionary constraint at the tissue level does not

apply to the primary structure of extracellular matrix proteins that control

the mineralization process. By extrapolating these results, we can assume

that the evolutionary constraint is maybe more eVective at the secondary or

tertiary structure levels, or even at the supramolecular level.

In a paper, Jackson et al. (2006) screened all the transcripts expressed

during shell calcification of the abalone, H. asinina, in particular the tran-

scripts encoding secreted proteins. Surprisingly, they found out that 85% of

the secreted proteins are unknown. A comparative scan between the obtained

EST sequences and the genome of the patellogastropod, Lottia scutum,

showed that only 19% of the secreted proteins of H. asinina have their

homologue in L. scutum, which constitutes another surprise. One of the

main conclusions of this study is that the shell is constructed from a rapidly

evolving secretome.

Another strategy was employed by Sarashina et al. (2006) to test the

antiquity of dermatopontin, the shell proteins of the freshwater snail

B. g labrata , charact erize d by Marxen et al. (2003b). They obt ained the hom o-

logues ofB. glabrata dermatopontin in seven other freshwater and land snails,

260 Marin et al.

with degenerate primers based on the dermatopontin amino acid sequence.

The dermatopontin gene is ancient because of its widespread repartition in

several metazoan lineages and because of its general function in extracellular

matrix assembly. However, the reconstruction of the phylogeny of molluscan

dermatopontins demonstrates that the recruitment of dermatopontin as a

shell matrix protein occurred twice independently in the two tested gastropod

lineages, between Carboniferous andTertiary. Although counterintuitive and

antiparsimonious, a scenario of two independent recruitments may empha-

size that the evolutionary constraints do probably not work at the level of the

genes encoding shell proteins but somewhere upstream. Alternatively, it is

also possible that the recruitment of the dermatopontin gene for calcification

was ancient, and abandoned later by many molluscan lineages (pseudogenes)

and conserved in others. In the future, this hypothesis should be confirmed by

investigating the presence of dermatopontin‐like proteins in the shell of

several mollusks, including bivalves and cephalopods, not only modern

most derived crossed‐lamellar gastropods.

At last, loose evolutionary constraints may also apply to the very acidic

shell proteins that are supposed to bind calcium carbonate crystals with a high

aYnity. Loose constraints can be tested experimentally with combinatorial

phage display libraries. These commercially available libraries consist of a

population of bacteriophages, genetically engineered to carry peptides located

at the end of one of the virus coat protein. For short peptides (typically seven

residues), the library contains all the possible peptides combinations with the

usual 20 amino acids. The library can be put in contact with mineral surfaces.

By applying rinsing steps of increasing stringency, the phages that exhibit the

highest aYnity with the mineral surface are selected and can be amplified and

sequenced. Few rounds of adsorption‐wash‐amplification permit the selec-

tion of the most strongly bound peptides. Experiments of this nature per-

formed on calcium carbonate surfaces gave interesting results (Belcher and

Gooch, 2000). In particular, they showed that several possible peptides with

very diVerent sequences could bind calcite or aragonite surfaces with a good

aYnity. This suggests that the mineral‐interacting domains on shell proteins

may be not particularly constrained and may be susceptible of amino acid

substitutions, which do not aVect their surface‐binding ability as long as the

crystal‐binding motifs of two to three amino acids are conserved.

D. Long‐Term Evolution of Shell Matrices and Microstructures:The Bivalve Example

Clearly, all the data taken together suggest that the molluscan shell secretory

repertoire has certain ‘‘plasticity’’and ‘‘evolvability’’ from group to group.

The impression is that a subtle balance is exerted between the plasticity of the


primary structures of the terminal products, the shell proteins, and evolu-

tionary constraints that operate at upstream level and keep the durability of

the microstructures, for example.

The complex relationships between shell microstructures and their asso-

ciated matrix call for further developments. In particular in bivalves, the shell

displays a certain number of microstructures, organized in layers, from two

to four. Shell microstructures are recognized as one of the parameters poten-

tially used in paleontology (after dentition, ligament insertion, adductor

scars, pallial line, and shell shape) for classifying fossil bivalves (Skelton,

1985). Bivalve shell microstructures were extensively described by Boggild

(1930), Kobayashi (1964), Oberling (1964), Taylor et al. (1969, 1973), Carter

(1980), Carter and Clark (1985), Shimamoto (1986), Carter (1990), and

others. Attempts were made to sketch some evolutionary trends in bivalve

microstructures, in particular by Taylor (1973), Carter (1980), Kobayashi

(1980, 1991), Uozumi and Suzuki (1981), Shimamoto (1993) but the task is

singularly diYcult. First, similar microstructures may appear in taxa, which

are widely separated in the phylogenetic tree, while they are absent in taxa of

intermediate position. The best example is the crossed‐lamellar structure

found in arcoid and veneroid bivalves. A second source of confusion is the

fact that the sequence by which these microstructures are associated in the

shell varies in the diVerent taxa: only in the extant venerid family, 12 combi-

nations were distinguished by Shimamoto (1986), while Uozumi and Suzuki

(1981) listed 47 combinations occurring in the bivalve class as a whole.

Clearly, the bivalve shell microstructures are the result of a mosaic evolution,

and adaptive convergences recur at diVerent taxonomic levels (Carter and

Clark, 1985; Taylor, 1973). In term of matrix plasticity, one can wonder

whether similar (in their shape) microstructures can be produced by very

diVerent shell matrices, or conversely, whether diVerent microstructures are

produced by very similar repertoires of proteins.

This question can be tackled in terms of energetic cost. Two decades ago,

Palmer (1983, 1992) calculated the cost of calcification in diverse mollusks.

He observed that the heaviest energetic cost for a mollusk corresponded

to the synthesis of the matrix and not the deposition of the mineral phase.

He concluded that mollusks that have a high content of organic matrix in their

shell were evolutionarily disadvantaged in comparison to those with a low

content. This remarkable finding, which had passed largely unnoticed, can be

integrated in the context of the long‐term evolution of bivalve microstructures.

Nacro‐prismatic textures exhibit a high content of organic matrix (up to 4–5%

of the shell weight, most of which is highly crossed‐linked and insoluble) and

are therefore considered as ‘‘primitive’’ (Carter, 1980; Taylor, 1973). On the

other side, crossed‐lamellar/homogeneous textures have a much lower shell

matrix content (below 0.5%), most of which is soluble. Although crossed‐lamellar microstructures appeared early in bivalves (see the lucinid family),

262 Marin et al.

most of the veneroid families (‘‘modern’’ heterodont bivalves), which all exhibit

combinations of crossed‐lamellar/homogeneous microstructures, appeared in

the Mesozoic times, and knew a rapid post‐Jurassic expansion, becoming

consequently the dominant bivalves (in terms of taxonomic diversity) in the

today’s oceans (Carter, 1980). The late but rapid burst of veneroid crossed‐lamellar bivalves suggests that they ‘‘invented’’ a new shell matrix repertoire for

controlling shell biomineralization and texture, a novelty, which gave them an

unquestionable evolutionary advantage.

At the microstructural level, such a possibility had been considered, in

particular in the dichotomic phylogeny of bivalve microstructures proposed

by Uozumi and Suzuki (1981). At the matrix level, putative diVerences in the

matrices associated with crossed‐lamellar and noncrossed‐lamellar bivalves

were evidenced by serological comparisons performed with diVerent shell

matrix antibodies (Muyzer et al., 1984; Marin, unpublished data). Clearly,

this needs to be tested now, by EST techniques, which would allow overall

comparisons of the secretory repertoires. We predict that shell matrices can

be represented as single points in a multidimensional space, where micro-

structures would represent strange attractors (Sprott, 1993). Such a repre-

sentation would conciliate the apparent plasticity of shell matrix proteins and

the evolutionary constraints at the microstructural level.

VI. Concluding Remarks

Sketching some long‐term perspectives for the future in biomineralization

research is by many aspects risky and presumptuous but forces us to look

back in the past and consider the evolution of the discipline over the last

decades. Forty years ago, the problem of the characterization of the mollus-

can shell matrix was mainly tackled at amino acid level, and the literature

dealing with amino acid compositions is abundant (Gregoire, 1972). At that

time, this level of analysis was discriminant enough for microstructural,

phylogenetic, or environmental purposes. However, it did not give any

chance to understand the dynamic of the shell formation process. In the

early 1970s, the discovery of the soluble matrix opened new biochemical

opportunities by investigating the shell matrix at the protein level, in spite

of certain technical limitations, inherent in the shell matrix itself. In the

1990s, the introduction of molecular biology techniques reinforced the ten-

dency to work at the protein level, as, at the same time, it opened perspectives

to characterize matrix constituents at the transcriptional level. We are still

in this phase, but are clearly moving toward the upper ‘‘ome’’ level of

analys is, that is, secretome (Jac kson et al., 2006) and proteom e (Be doue t

et al., 2007), which drives us to supramolecular chemistry and hierarchy in

biomineralization. Interestingly, these upper levels of analysis bring us


back to the phy siology of the calcifyi ng mantl e tissue, one aspect that had

been neglect ed for years. Does it mean that we have now acquire d a bette r

unde rstand ing on how a mo llusk makes its shell? For some aspect s, the

answ er is clearly yes. For some others , our ignoran ce is abyssal ,

consider ing the size of the phy lum Mo llusca, an d the few studied specie s.

The general impr essio n is that the more biologi cal models we study, the more

matrix pro teins we find. Enum erating precisely the protei ns is a necessa ry

requir ed step for draw ing the outli ne of the di Verent functi onal domains and

protei n families, for phylogenet ic pur poses, but, once again, an exhaust ive

catalo g of the shell protei ns will not help so much in unde rstan ding the shell

form ation. We are confront ed to the limit s of a reductio nist approach , which

consis ts in exp laining one given physica l propert y (like the polymor ph

selec tion) by the presence of one or two matr ix consti tuents. She ll matrices ,

when consider ed as biologi cal object s, exhibi t ‘‘eme rging propert ies’’ that are

much more than the sum of their parts , matr ix protei ns. Future attempts to

apply revers e geneti cs (gene knock down with morpholi no oligos ) on mollu sk

larva e, accu rate qua ntification of the di Verent transcript s levels in distinctparts of the calci fying mantle, and finally extended genome ‐ to ‐ genome

compari sons may allow filli ng pa rtially the gap. In parti cular, these

appro aches sh ould permit to relat e the struc ture of moll uscan shell protei ns

to their function s, to unv eil the spati otempor al express ion of the secret ory

sequen ce, and at last establ ish the secretory repert oire an d propose

phylogen ies of calci fying protei ns. However, serio us conceptual e Vorts willadditi onally be require d for explaining how these beauti fully orga nized shell

biomi nerals like na cre or prisms emerge from liqui d syst ems.

Acknowledgme nts

This chapter is a contribution to an ‘‘Aide Concerte e Incitative Jeunes Chercheurs’’ (ACI JC

3049) awarded to F. M. by the French ‘‘Ministe re De le gue a la Recherche et aux Nouvelles

Technologies’’ for the period 2003–2006. For the period 2007–2010, this work is supported by an

ANR project (ACCRO–Earth, ref. BLAN06–2_159971, coordinator Gilles Ramstein, LSCE,

Gif/Yvette). In 2004, the ‘‘Conseil Regional de Bourgogne’’ (Dijon, France) provided financial

supports for the acquisition of new equipment in Biogeosciences research unit. F.M. thanks

Claudie Josse (Laboratoire de Reactivite des Solides, UB, Dijon) for her help in handling SEM,

and EGIDE (PAI Cogito 09084XG) for promoting the collaboration with Professor D.M. At

last, but not least, F.M. thanks Alain Godon for his kind help in redrawing Figs. 2 and 3.

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Molluscan Shell Proteins: Primary Structure, Origin, and Evolution

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