Soluble Organic Matrices of the Calcitic Prismatic Shell Layers ofTwo Pteriomorphid BivalvesPINNA NOBILIS AND PINCTADA MARGARITIFERA*

Received for publication, May 6, 2002, and in revised form, February 7, 2003Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M204375200

Yannicke Dauphin‡

From the From Laboratoire de Paleontologie, FRE 2566, Universite Paris XI-Orsay, F-91405 Orsay, France

The calcitic prisms of the shells of two bivalves, Pinnaand Pinctada, are considered simple prisms accordingto some morphological and mineralogical characteris-tics. Scanning electron microscopic and atomic forcemicroscopic studies show that the microstructures andnanostructures of these two shells are different. Pinnaprisms are monocrystalline, whereas Pinctada prismsare not. Moreover, intraprismatic membranes are pres-ent only in the Pinctada prisms. The soluble organicmatrices extracted from these prisms are acidic, buttheir bulk compositions differ. Ultraviolet and infraredspectrometries, fluorescence, high pressure liquid chro-matography, and electrophoresis show that the sugar-protein ratios and the molecular weights are different.Sulfur is mainly associated with acidic sulfated sugars,not with amino acids, and the role of acidic sulfatedsugars is still underestimated. Thus, the simple prismconcept is not a relevant model for the biomineraliza-tion processes in the calcitic prismatic layer of molluskshells.

Among more than 60 known different biominerals, calciumminerals are the most abundant. These exoskeletons (alsocalled hard tissues or shells) have morphological, chemical, andphysical properties that are never shown by the correspondingabiotic crystals. They are biocomposites (bioceramics), and or-ganic macromolecules are their key components. Much of whatis known about biominerals is deduced from the nacreous lay-ers of mollusks, and it is believed that the organic matrixserves as a guide for crystal growth (1, 2). All of the SOM1

extracted from mollusk shells are highly acidic as shown bytheir amino acid contents or by ion exchange chromatography(3–5). Unfortunately, it is often said that a mollusk shell con-sists of an outer calcitic and an inner aragonitic layer (nacre),although this structure is rare. In fact, these shells exhibitvarious microstructural types, the arrangement of whichstrongly depends on the taxa. Currently, little data are avail-able on the organic matrices extracted from mollusk shells.

Pinna nobilis and Pinctada margaritifera are two modern

pteriomorphid bivalves with shells composed of an outer cal-citic prismatic layer and an inner aragonitic nacreous layer.Their calcitic prisms offer remarkable advantages to investi-gate the organization of biocrystals. For example, they arelarge units that globally exhibit a single crystal-like organiza-tion. These prisms are built by a series of growth steps andsurrounded by thick organic walls (6–10). According to Tayloret al. (11) the outer layers of Pinna and Pinctada are composedof calcitic “simple prisms.” Their crystallographic c axes arenormal to the layer surfaces. Illustrations of a transverse sec-tion of the prisms of Pinctada by Wise (12) are similar to thoseof Taylor et al. (11) and follow the simple prism concept. Thesecalcitic layers have high S and magnesium contents (13–16).Amino acid studies of the soluble intraprismatic matrices ofboth genera have shown high contents of aspartic acid, glycine,alanine, and glutamic acid (17, 18). However, repeated obser-vations have shown some differences between the two shells. InPinctada martensi, thin sections observed in polarized lightshow that each prism extinguishes in several smaller blocks (8,19). Wada (9) observed uniaxial crystals and partial irregularextinctions in horizontal sections under crossed nicols. Theindividual prism of Pinna exhibits a monocrystalline extinction(20, 21).

Dissolution of the crystal units releases various macromole-cules into solution, and it is well known that organic compo-nents are key participants in the control processes of shape andstructure in mineralized tissues (2, 3, 17, 22). Only some pro-teins have been isolated from the aragonitic nacreous layers ofPinctada (23) since the first evidence of the presence of organicmatrices (24). Thus, before the cloning of “pure” proteins ofmollusk shell layers, a better knowledge of the bulk composi-tion of their SOM is necessary as a first step in understandingthe mechanisms of layer formation. From preliminary studiesof the molecular weights of the SOM extracted from the prisms,Pinna and Pinctada are different (15, 25, 26). While pursuing along term study of calcified biominerals, the present study wasundertaken to extract and compare the SOM of the calciticprisms of Pinna and Pinctada and to relate these SOM to thesimple prism concept. Unlike many studies, this work is notfocused on only the protein content. The location of the organicmatrices and the presence of sugars are also considered.

EXPERIMENTAL PROCEDURES

Materials—Specimens of P. margaritifera (L.) (Pteriomorpha, Pteri-oida, Pteriacea, Pteriidae) were collected in French Polynesia. P. nobilisL. (Pteriomorpha, Mytiloida, Pinnacea, Pinnidae) samples came fromthe Mediterranean Sea.

Standards—Isoelectric focusing calibration kit (pH 2.5–6.5) wasfrom Amersham Biosciences. Low range SDS-PAGE standards (19–107kDa), kaleidoscope-prestained standards (6.9–202 kDa), and gel filtra-tion standards were from Bio-Rad. Chondroitin sulfate A was pur-chased from Sigma. Rapid Decalcifier was from Apex Engineering Prod-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom correspondence should be addressed: Laboratoire de Pa-leontologie, bat. 504, Universite Paris XI-Orsay, F-91405 Orsay,France. E-mail: dauphin@geol.u-psud.fr.

1 The abbreviations used are: SOM, soluble organic matrix/matrices;SEM, scanning electron microscope; AFM, atomic force microscope;HPLC: high pressure liquid chromatography; FTIR, Fourier transforminfrared spectrometry; IEF, isoelectric focussing electrophoresis; IPG,Precast Immobilin DryStrip; BSA: bovine serum albumin; CS, chon-droitin sulfate; XANES, x-ray absorption near-edge structure.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 17, Issue of April 25, pp. 15168–15177, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org15168

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

ucts Corporation. BSA was from Eurobio, and chymotrypsin was fromMerck.

Scanning Electron Microscopy (SEM)-Atomic Force Microscope(AFM)—The traditional method for obtaining information about theshell microstructures is scanning electron microscopy. Fractures andpolished etched sections have been observed with Philips 505 and XL30SEM. Acidic and enzymatic etchings were used to reveal the details ofthe microstructures of the polished sections. Samples were also studiedusing a Nanoscope IIIa (Digital Veeco) multi-mode scanning probemicroscope operating in tapping mode. The tapping mode AFM utilizesan oscillating tip at a tip amplitude of approximately several tens ofnanometers when the tip is not in contact with the surface. Because thetip is no longer in permanent contact with the sample surface duringthe scanning motion, sample alteration can be avoided. The resolutionof tapping mode AFM is on the order of a few nanometers. Details of theetchings for SEM and AFM observations are given in the figure legends.

Extraction and Purification of the Soluble Organic Matrix—Sampleswere immersed in 3% NaClO for 1 h to remove organic contaminants,rinsed with Milli-Q water, dried, and ground into powder. Powderedsamples were immersed in 5 ml of Milli-Q water and then decalcified byprogressive addition of 50% acetic acid so that the pH (automaticallycontrolled with a titrimeter) is above 4. The entire extract was directlycentrifuged at 21,000 � g for 15 min, which separated the supernatant(soluble) and precipitated (insoluble) fractions. The soluble fraction wasdesalted by exchange with Milli-Q water on a Microconcentrator (Fil-tron) using a 3-kDa cut-off membrane and lyophilized.

Fluorescence-UV Spectrometry—SOM were dissolved in Milli-Q wa-ter. Fluorescence spectra were recorded on a PerkinElmer LS40 fluo-rescence detector equipped with a Xenon lamp and a silica flow cell.Sample spectra were corrected for background to minimize residualfluorescence effects. UV spectra were recorded on a Shimadzu UV-1601spectrophotometer equipped with deuterium and tungsten lamps anddouble beam optics. The monochromator slit aperture is fixed at 2 nm.Before a sample spectrum was run, a background spectrum was meas-ured for Milli-Q water. All spectra were recorded from 250 to 350 nm.

Infrared Spectrometry—All spectra were recorded at a 4-cm�1 reso-lution with 64 scans with a strong Norton-Beer apodization on aPerkinElmer Model 1600 Fourier transform infrared spectrometer inthe wave number range of 4000–450 cm�1. The spectrometer wasequipped with a diffuse reflectance accessory, which permits DRIFTmeasurements with high sensitivity on powders. All spectra were cor-rected by the Kubelka-Munk function, and the bands were identified bythe software. The system was purged and permanently maintainedunder nitrogen to reduce atmospheric CO2 and H2O absorption. Abackground spectrum was measured for pure KBr. The ratio of thesample spectra was automatically determined against background tominimize CO2 and H2O bands. Several spectra from the same extractedSOM and from different SOM were done with correlation coefficientshigher than 95%. Only minor differences attributed to humidity aresometimes present.

HPLC—Chromatographic analyses were performed using two TSKG5000PW and G3000PW columns connected in series (200–1 � 106 Da).They were eluted with 0.2 mM Tris, pH 7.5, at a flow rate of 1 ml/min inhigh sensitivity refractive index detector PE 200 (PerkinElmer LifeSciences) and thermo separation products detector 4100 and monitoredat 226, 254, and 278 nm. These two detectors were connected in seriesso that there is a difference in the elution times.

Other analyses were done using the same TSK columns connected inseries, eluted with a dissociative buffer (4 M guanidinium chloride, pH6, at a flow rate of 0.70 ml/min), and monitored at 275 nm. SOM werealso chromatographed using a Superose 12 column (APB) with a sepa-ration range from 1 to 300 kDa and exclusion limit of 2000 kDa. TheSuperose 12 column was eluted with 4 M guanidinium chloride, pH 6, ata flow rate of 0.70 ml/min and monitored at 275 nm. The lyophilizedSOM were dissolved in the buffer overnight, and all analyses were doneat ambient temperature.

Isoelectric Focusing Electrophoresis (IEF)—Isoelectric points weredetermined according to Dauphin and Cuif (16, 27). One-dimensionalmicroslab isoelectric focusing was done on homogeneous polyacryl-amide gels. With the Bio-Rad Model 111 Mini IEF cell, the gel was runwithout electrode buffers (non-denaturing dry IEF). The lyophilizedSOM were redissolved in Milli-Q water. Focusing was carried out in astepped fashion to prevent overheating.

Two staining procedures were used: 1) Coomassie Blue-Crocein Scar-let for proteins or weakly glycosylated proteins and 2) Acridine Orangefor acidic sulfated sugars. For the Coomassie Blue method, gels wereplaced in 27% ethanol, 10% acetic acid, 0.04% Coomassie Blue R-250,0.5% CuSO4, and 0.05% Crocein Scarlet for 1.5 h (fixing and staining

solution). The first destaining solution contained 12% ethanol, 7% ace-tic acid, and 0.5% CuSO4. The second destaining solution contained 25%ethanol and 7% acetic acid. For the Acridine Orange method, gels wereplaced in a solution of methanol (40%) and acetic acid (10%) for 30 min(fixing solution). Acridine Orange was dissolved in Milli-Q water (20 mgof Acridine Orange in 95 ml of H2O and 5 ml of methanol). Gels wereplaced in the solution for 30 min and then destained with Milli-Q water.

Markers—The low pI calibration kit (APB) with pH among 2.5 and6.5, BSA, and ribonuclease were used. They were stained with Coomas-sie Blue R-250.

Two-dimensional Gel Electrophoresis—The molecular groups wereseparated in the first dimension by isoelectric focusing in IPG strips(Precast Immobilin DryStrip, 11 cm, pH 3–10, APB) on a Multiphor IIsystem. The lyophilized samples were dissolved in the sample buffer (8M urea, 0.5% Triton X-100, 40 mM dithiothreitol, Pharmalyte 3–10,bromphenol blue) and centrifuged for 10 min at 17,500 � g. Thissolution was applied to the Immobilin strips in a horizontal reswellingcassette overnight. The settings used for IEF were 300 V for 6.5 h and2000 V for 11 h. The Multitemp III cooling bath was set at 20 °C.

After the focusing was complete, the second step was done accordingto Dauphin (28). After electrophoresis, the gels were placed in 40%methanol with 10% acetic acid for 24 h to fix the proteins and to removethe SDS. Proteins and glycoproteins were detected by silver staining(Bio-Rad silver stain kit), and sulfated acidic mucopolysaccharides werestained with Alcian Blue. Silver staining was followed by Alcian Blue.

XANES—The work was carried out at the European SynchrotronRadiation Facility. The ID21 Scanning x-ray Microscope uses Fresnelzone plates as focusing optics to generate a submicron x-ray probe,which is used to investigate the sample with various contrast mecha-nisms (fluorescence, transmission, and phase contrast). An energy-dispersive high purity Ge detector (Gamma-Tech, Princeton, NJ)mounted in the horizontal plane perpendicular to the beam collects thefluorescence emission photons. This geometry minimizes the contribu-tion of elastic scattering. Provided the sample is thin enough, a Siphotodiode can be mounted downstream from the sample to exploit thetransmission signal as well. An energy range between 2 and 7 keV isavailable, which gives access to the K-edge of sulfur at 2472 eV. Theenergy scan is ensured by a fixed-exit double crystal Si111 monochro-mator located upstream from the microscope, which offers an energyresolution necessary to resolve XANES features. This experiment re-quired operating the x-ray microscope under vacuum to avoid the strongabsorption of the sulfur emission lines by air.

RESULTS

Microstructure and Nanostructures

Untreated longitudinal fractures in the outer layer of P. no-bilis do not exhibit intraprismatic structures. Only longstraight parallel prisms are visible (diameter 70 �m) (Fig. 1A).Rare and faint growth lines are sometimes seen on the outersurface of the prisms in such fractures. A polished and stronglyetched transverse section shows the thick organic interpris-matic walls and the polygonal shape of the prisms (Fig. 1B).Polished and etched longitudinal sections show the thick inter-prismatic walls and transverse synchronous growth linesacross the adjacent prisms (Fig. 1C). Polished, fixed, and de-calcified transverse sections show intraprismatic parallelcrests (Fig. 1D). The interprismatic wall is somewhat etched byenzymatic hydrolysis, which supports its organic composition,and the intraprismatic parallel crests remain visible (Fig. 1E).Thin sections observed with cross-nicols show that each prismis a monocrystal with a unique extinction pattern (Fig. 1F).However, these prisms are not compact structures. They arecomposed of oblique and elongated crystallites (Fig. 1, G–H),the width of which varies from 150 to 180 nm. These crystal-lites are subdivided into smaller rounded units with distinctboundaries in height and phase images, suggesting that theyare surrounded by organic envelopes (Fig. 1I).

Similar unetched fractures of Pinctada show another pat-tern. The elongated prisms are not so straight (Fig. 2A). Theirsurfaces seem to be corrugated, and pieces of organic sheathscut along the growth lines are preserved (Fig. 2B). The innerstructures of the prisms seem homogeneous in transverse-un-treated sections, and after complete removal of the mineral

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves 15169

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

portion, only the outer thick organic walls are present. How-ever, the polished and etched transverse sections are differentfrom those of Pinna. Enzymatic hydrolyses reveal intrapris-matic sinuous lacunae (Fig. 2C). The nonspecific enzymes usedand the basic pH suggested that these lacunae are the remainsof intraprismatic organic membranes. The orientations of crys-tallites on both sides of these intraprismatic membranes withina prism are not similar (Fig. 2D). These sinuous intraprismaticlacunae are also present in fixed and etched transverse sections(Fig. 2E). In such preparations, the interprismatic walls arepartially destroyed. Transverse thin sections observed withcross-nicols confirm that the prisms are composite crystals

(Fig. 2F). AFM observations show that the elongated crystal-lites are irregularly aligned (Fig. 2, G and H). Their width is�110 nm, whereas their length varies from 250 to 400 nm.Round units at 50–70 nm in diameter are sometimes present.As with Pinna, the crystallites seem surrounded by a thinorganic layer according to the phase images (Fig. 2I).

The long polygonal calcitic prisms in both Pinna andPinctada are surrounded by a thick organic wall and exhibitgrowth lines in longitudinal sections. However, prismatic unitsof Pinna are monocrystalline, whereas those of Pinctada arenot, as shown by thin sections and complex preparative pro-cesses. The enzymatic hydrolyses were done at pH �7 so that

FIG. 1. Microstructures and nano-structures of the calcitic prisms ofP. nobilis. A, vertical fracture in theouter layer showing the long parallel reg-ular prismatic units. No pattern is visibleon the outer surface of the prisms. B,transverse polished, fixed, and etched sec-tion showing the thick organic walls (w)(6% glutaraldehyde, 1 h; Rapid Decalci-fier, 30 min). C, vertical polished andetched section showing the thick inter-prismatic walls (w) and the regulargrowth lines. D, transverse polished,fixed, and etched section showing the in-terprismatic wall (w) and a pattern of par-allel crests. Glutaraldehyde � formic acid� Alcian Blue solution. E, inner surfaceshowing the same pattern of parallelcrests and the walls (w) (pronase in Trisbuffer, pH 8, at 30 °C for 3 h). F, thinsection observed in transmitted light(cross-nicols) showing the walls (w) andthe monocrystalline extinction of eachprismatic unit. G, vertical polished andetched section showing the aligned acicu-lar crystallites (AFM tapping mode; 2%glutaraldehyde, 0.1% formic acid, 10 s).H, detail of the same. I, tranverse pol-ished and etched section of the prismsshowing the small crystallites surroundedby an organic thin layer (AFM imagephase; 2% glutaraldehyde, 0.1% formicacid, 10 s).

FIG. 2. Microstructures and nano-structures of the calcitic prisms ofP. margaritifera. A, vertical fracture inthe outer layer showing the long parallelirregular prismatic units. B, detail of thesame showing pieces of the outer wallstripped off along the transverse growthlines. C, polished and etched prismsshowing the differential behavior of theouter walls (w) and the sinuous intrapris-matic organic membranes (arrows) de-stroyed by proteolysis (Alcalase, 25 h,38 °C). D, detail of the same showing thechange in crystallite orientation on bothsides of an intraprismatic organic mem-brane seen as a lacuna after proteolysis(arrow). E, transverse polished, fixed, andetched section showing the partly de-stroyed interprismatic wall (w) and intra-prismatic sinous lacunae (arrows). Glutar-aldehyde � formic acid � Alcian Bluesolution. F, thin section observed in trans-mitted light (cross-nicols) showing thewalls (w) and the composite structure (ar-rows) of the prismatic units. G, tranversepolished and etched section showing thecrystallites, the alignment of which is notregular (AFM scan field 5 mm; 2% glutar-aldehyde, 0.1% formic acid, 10 s). H, detailof the same (image phase). I, detail of thesame showing elongated crystallites.

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves15170

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

the mineral part is not strongly etched and only a small disso-lution can occur. Thus, the intraprismatic sinuous lacunae inPinctada indicate the presence of sinuous organic membranes.Besides, there is a clear difference between these sinuous in-traprismatic organic membranes and the outer walls. The sin-uous intraprismatic organic membranes are destroyed by acidand enzymes, whereas the outer walls are not. The ultrastruc-ture of Pinctada is then more complex than that of Pinna,which suggests that the intraprismatic organic matrix ofPinctada is also more complex. Hence, the simple prism con-cept appears not to be appropriate.

Bulk Composition

The emission scan of CS exhibits a small peak at 287 nm anda main peak at 353 nm for a 257-nm excitation. Peaks are at290 and 342 nm for BSA (Fig. 3A). Only a small peak is visibleat 287 nm in Pinctada and a shoulder is visible in Pinna. Theemission scan of CS shows a shoulder at 310 nm and a mainpeak at 353 nm for a 275-nm excitation. A peak at 342 nm isvisible in the BSA spectrum. A small peak at 303 nm ispresent in Pinna and Pinctada SOM (Fig. 3B). CS shows apeak at 353 nm for a 287-nm excitation, whereas that of BSAis at 342 nm. Only shoulders are present in Pinna andPinctada spectra (Fig. 3C). From UV spectra, it appears thatPinna and Pinctada SOM are not pure proteins and thatthese proteins have a low content of aromatic amino acids(data not shown). From fluorescence and UV spectra (datanot shown), it may be inferred (1) that the SOM of Pinna andPinctada are different (2), that these matrices are not pureproteins (3), and that their aromatic amino acid contents arevery low.

In infrared spectra, the amide I band is the most intenseabsorption band for proteins. The presence of bands arisingfrom amino acid side chains in the region between 1800 and1400 cm�1 (amides I and II) has been thoroughly investigated(29). It has been established that among the 20 proteinogenousamino acids, only residues arginine, asparagine, glutamine,aspartic and glutamic acids, lysine, tyrosine, histidine, andphenylalanine have intense absorption in this region. Pinnahas nine bands, and Pinctada has eight bands in this part ofthe spectrum. The prominent band in the two matrices is theamide I band near 1653 cm�1; thus, it may be supposed thatcomponents have adopted the �-helical conformation (Figs. 4and 5). However, precise interpretations of bands in the amideI region are difficult, because there is an overlap of the �-helicalwith random coil structures. The 1647 cm�1 band (amide I)

may be assigned to unordered structures, this band being ab-sent from Pinctada. Other bands show the presence of �-turnand �-sheet structures. The prominent amide II bands are near1560 cm�1 (Pinna) and 1540 cm�1 (Pinctada). Strong carbox-ylate absorption bands are present at 1419–1420 and 1717cm�1 in both SOM. Bands at 1717 and 1575 cm�1 are usuallyassigned to aspartic acid, whereas bands at 1712 and 1558cm�1 are assigned to glutamic acid. These four bands arepresent in Pinctada, whereas bands corresponding to aspartate(1622 and 1678 cm�1) and glutamate (1610 and 1670 cm�1) areabsent. In Pinna, bands assigned to aspartic acid (1574 and1717 cm�1) are present, but bands related to aspartate areabsent. One band is present for glutamic acid (1560 cm�1), andtwo bands are present for glutamate. The small band near 1245cm�1 may imply that some molecules are sulfated (Fig. 5).Amide A bands are similar in both samples and are not specificfor secondary structures.

All of these bands are present in both proteins and sugars.Thus, it is possible that the strong amide A, I and II bands, arealso due to sugars as shown by the spectra of CS (30, 31). Onthe other hand, bands between 1000 and 1150 cm�1, which areabsent from protein spectra, are usually considered character-istic of only the sugars. Thus, in complex components, it is

FIG. 4. FTIR spectra of the SOM of Pinna and Pinctada.

FIG. 3. Fluorescence spectra of two standards, BSA (protein) and CS (acidic sulfated sugar), and of the SOM of Pinna andPinctada showing that the SOM are not pure proteins. Excitation: 257 nm (A), 275 nm (B), 287 nm (C).

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves 15171

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

difficult to infer the low or high contents of sugars from theintensity of bands of only the 1000–1150-cm�1 region. In bothSOM, these specific bands are weak, but this is not necessarilyevidence of a low sugar content. Moreover, some moleculesseem sulfated.

Molecular Masses

The HPLC chromatogram of Pinna at 226 nm in non-disso-ciative buffer (Tris) shows a small peak of excluded large mol-ecules (�1 � 106 Da), a main peak at 277 kDa, and a minorpeak estimated at 12 kDa (Fig. 6A). At 254 and 278 nm, theoverall peaks are similar to those detected at 226 nm, but theexcluded molecules are the main peak. Only the largest andsmallest molecular masses are present in the refractometricchromatogram, the main peak being at 277 kDa (Fig. 7). Thereis a strong decrease in the intensity of the peaks from 226 to278 nm, showing a low protein content. Refractive index detec-tors (or refractometers) are sensitive to a wide range of organiccompounds, but they are typically used to analyze compoundsthat do not have strong absorbance in the UV range such assugars. In the experimental conditions used, low quality com-mercial BSA shows one or two small peaks, whereas highpurity globular proteins do not show peaks. CS shows a strongpeak. These data may imply that the main components of thePinna SOM are sugars or highly glycosylated proteins.

The 226-nm profile of Pinctada shows three distinct peaks ofincreasing intensities: one of excluded molecules, a second oneat 377 kDa, and the last one at 110 kDa (Fig. 6B). As for Pinna,the 278- and 254-nm profiles are similar to each other anddifferent from the profile at 226 nm. The main peak is at 110kDa, the middle peak is a shoulder, and the excluded moleculesare present. These three peaks are present in the refractomet-ric profiles (Fig. 7), the excluded molecules being the mostintense. It should be noted that in Figs. 6 and 7, elution timesare different for the equivalent peaks in each elution profilebecause of the serial connection of the two detectors.

The comparison of the UV and refractometric HPLC profilesconfirms the UV-fluorescence data. The SOM of Pinna and

FIG. 6. HPLC UV profiles of Pinna (A) and Pinctada (B) show-ing the composite nature and the different molecular weights ofthe SOM. Non-dissociative buffer (Tris).

FIG. 5. Detailed FTIR spectra of the SOM of Pinna and Pinctada showing the different bands in the amide I, amide II, and thesugar region (A) and in the amide I and II domains (B).

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves15172

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Pinctada are different. Not only are the molecular weightsdifferent, but each peak has a peculiar composition. The226-nm profiles of Pinna and Pinctada show similar intensi-ties, but the refractometric profile of Pinna is more intensethan that of Pinctada. UV and refractometric profiles of asample were acquired during the same elution, so that theirratios are not dependent of the quantity of SOM. According tothe comparison of these profiles and the 226/278 nm ratios,Pinna seems to contain a higher proportion of sugars thanPinctada.

Components with molecular masses greater than 106 Da arepresent in Pinna (Fig. 8A), the main peak being at �120 kDa.The Pinctada profile shows more peaks. A faint shoulder isindicative of excluded components (�106 Da), other peaks be-ing at �98 and 23 kDa (Fig. 8A). The separation range ofSuperose 12 chromatography in the dissociative buffer (1,000–300,000 Da) is narrower than that of the TSK columns. ThePinna profile shows an excluded peak (�300 kDa) and a broadpeak at 34 kDa (Fig. 8B). The Pinctada profile is more complexwith a small excluded peak, several shoulders, a main peak ofapparent molecular mass of 20 kDa, and several small peaks(Fig. 8B). Clearly, there is a discrepancy in the molecular massestimated for the molecules in the major peak of the PinnaSOM on the two different columns (120 and 34 kDa). The basisfor this discrepancy is not readily apparent but may indicatesome peculiarity regarding the molecules contained in thispeak.

Despite the observed differences in the dissociative buffer,Pinna and Pinctada SOM are different. The comparison of the280-nm (non-dissociative buffer) and 275-nm (dissociativebuffer) profiles shows that the excluded peaks are less promi-nent in the dissociative buffer. There is a good correspondenceof the apparent molecular masses among the three profiles ofPinctada but not in Pinna. The different behavior of the twoSOM in the two buffers is consistent with the previous results.Some of the macromolecules of Pinna are poorly dissociated byguanidinium chloride, a behavior characteristic of highly gly-cosylated molecules. In contrast, Pinctada SOM is better dis-sociated and the sugar component is probably of lesserproportion.

Acidity

CS (an acidic and sulfated sugar) can be stained with Acri-dine Orange, but no discrete bands are present in this non-denaturing electrophoresis (Fig. 9A, center lane). Standard pro-teins are not stained with Acridine Orange but are stained withCoomassie Blue-Crocein Scarlet (Fig. 9A, left lane). The Pinna

SOM is successfully stained with Acridine Orange, showingthat the main part of the organic components consists of sul-fated sugars, the pI of which is lower than 5.8 (Fig. 9A). A faintCoomassie Blue-Crocein Scarlet stain indicates that the pro-tein components are slightly less acidic (Fig. 9A). Acidic sul-fated sugars are also stained by Acridine Orange in Pinctada,but the proteins stained with Coomassie Blue-Crocein Scarletare more acidic than those of Pinna (Fig. 9A). The two dyesused show that the SOM of Pinna and Pinctada are composedof proteins and acidic sulfated sugars. However, Pinna andPinctada SOM are different. The protein part of Pinctadaseems more acidic than that of Pinna.

Two-dimensional Electrophoretic Separation

Pinna—The Alcian Blue stain of the IPG strip shows a gra-dient from a light color (basic part) to an intense blue color atthe acidic end (Fig. 9B). Thus, it may be suggested that largemolecular mass components (� 300 kDa) do not penetrate intothe two-dimensional gel. These acidic components are stronglystained with Alcian Blue but not with silver; thus, it may beinferred that they are sulfated sugars. A very weak silver stainis visible only in the basic part of the IPG strip (Fig. 9B). Themain part of the two-dimensional gel is not stained, and onlythe low molecular masses-acidic pI are stained with AlcianBlue (Fig. 9B). There is a contrast between the acidic parts ofthe IPG strip and the two-dimensional gel. The strip is heavilyAlcian Blue-stained, whereas the two-dimensional gel is faintlystained. The silver stain is faint and visible only in the acidicpart of the two-dimensional gel. Despite the use of dissociativedenaturant buffers containing urea, SDS, dithiothreitol, 2-mer-captoethanol, and Triton X-100, there is no discrete band or

FIG. 7. HPLC refractometric profiles of Pinna and Pinctadashowing the high content of sugars in the SOM of Pinna.

FIG. 8. HPLC UV profiles at 275 nm of Pinna and Pinctada ina dissociative buffer (guanidinium chloride). A, profiles showingthe composite nature and the different molecular masses of the SOM.Same columns as in Fig. 6 (TSK columns). B, profiles in a Superose 12column.

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves 15173

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

spot, and according to the stain, the main part seems composedof acidic sulfated sugars.

Pinctada—The entire length of the IPG strip is heavilystained: the acidic part with Alcian Blue, indicative of sulfatedsugars, and the basic part with silver, indicative of proteins(Fig. 9B). The main part of the two-dimensional gel is silver-stained with a good separation according to pI but with nodistinct band according to molecular masses (Fig. 9B). Theacidic-low molecular mass part of the gel shows some unusualpatterns and is also Alcian Blue-stained. As for Pinna, it maybe suggested that a part of the acidic portion of the SOM iscomposed of molecular masses higher than 300 kDa, and thesecomponents do not penetrate into the two-dimensional gel. TheAlcian Blue-stained two-dimensional gel prior to the silverstain does not show a strong blue color (data not shown).

The striking feature of the two-dimensional gels is the ab-sence of the spotty pattern usually seen for soft tissue proteins,despite the use of dissociative buffers. Smears are usuallyindicative of a high degree of glycosylation. Pinna SOM seemsto be composed of acidic sulfated sugars, a part of which hasmolecular masses higher than 300 kDa. Pinctada SOM alsoshows components with molecular masses higher than 300kDa, but this SOM contains proteins and sulfated sugars witha large pI range as shown by the IPG strip. The lower molec-ular weight components of the SOM also seem to be composedof proteins with a larger range of molecular weights and pI.

Sulfur Contents—Reference spectra are in accordance withpublished data (32, 33). The S–K edge spectra of methionineand cysteine show a main peak at 2.473 keV (Fig. 10A). Cystinewith a disulfide bond shows a double peak in the same region(Fig. 10B). A similar double peak is also present in phenyldisulfides (data not shown). The sulfated sugar, CS, shows amain peak at 2.482 keV and no peak in the S amino acid region(Fig. 10B). The spectra of the SOM extracted from Pinna andPinctada also show a main peak at 2.482 keV, indicative of a

high sulfate content based on the CS spectrum (Fig. 11A). Theprofile of Pinctada shows a double peak similar to that ob-served with CS. Pinctada shows small peaks at 2.47 keV cor-responding to S amino acids according to the reference spectra,whereas the Pinna profile is flat for S amino acids. Theseresults are consistent with the HPLC and electrophoretic data.Pinctada SOM contains more proteins than that of Pinna.

SEM micrographs show sinuous intraprismatic membranesin Pinctada prisms (Fig. 2, C–F, arrows) but not in Pinna (Fig.1, D–F). In situ spectra of these sinuous intraprismatic mem-branes of Pinctada confirm that they are organic. A main peakat 2.48 keV and a very faint shoulder at 2.47 keV may beindicative of some S amino acids (Fig. 11B). The S amino acidregion is weaker in this spectrum than that of the extractedSOM. The detailed structures of the intraprismatic membranesare unknown, but as for membranes in other microstructuresin mollusk shells (i.e. the nacreous layer), they are probablycomposed of soluble and insoluble parts, the compositions ofwhich are different. It is conceivable that the difference be-tween the SOM spectrum and the in situ spectrum is due to theratio of soluble and insoluble matrices involved in the intra-prismatic membranes.

In both samples, XANES spectra show that the main sulfurspecies is sulfate, not S amino acids. However, the PinctadaSOM also contains some sulfur corresponding to amino acidswith a small double peak, which may suggest the presence ofcystine. However, the ratios of the three small peaks in theamino acid region as well as the energy of the third peak arenot the same in the spectra of cystine (Fig. 10B) and PinctadaSOM (Fig. 11A).

DISCUSSION

From the above results, three topics can be discussed: 1) themethods used, 2) a comparison with other calcitic prisms, and3) the relevance of the simple prism concept.

FIG. 9. A, non-denaturing IEF of the SOM of Pinna and Pinctada. Coomassie Blue-stained standard proteins and Acridine Orange-stained CSare shown. The sugar fractions of the SOM are more acidic than the proteins. B, two-dimensional gels. Pinna and Pinctada SOM are stained withAlcian Blue and then silver. Standard proteins are silver-stained. AO, Acridine Orange; BCC, Coomassie Blue-Crocein Scarlet.

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves15174

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Methods—Because EDTA tends to form non-removable com-plexes with organic materials extracted from bones or shells(34–36), a moderate acidic decalcification was used (pH 4). It issometimes said that acetic acid decalcification is a harsh proc-ess denaturing the soluble organic matrix, but the accuracy ofthis assessment is questionable. If the acetic acid decalcifica-tion hydrolyzes and cleaves the SOM into smaller molecules,low molecular weight peaks and bands should be present inHPLC and electrophoresis. The presence of large molecularweight species shows that the hydrolysis of the SOM during thedecalcification is moderate if not absent. These results areconsistent with the usual hypothesis saying that when thedecalcifying solution and organic components have similar pH,the organic matrices are not altered. Previous amino acid anal-yses of the SOM of mollusk shells have shown that the averagepI is near 4 (17, 18). Thus, it may be inferred that the decalci-fication used does not substantially alter the SOM. In addition,concentrated HCl has been used to extract the intraskeletalmacromolecules of ascidians and no major effect was observedon the extracted macromolecules (37).

UV spectrometry and fluorescence show that the SOM studiedhave very low aromatic amino acid contents. Although thesemethods are relatively simple, they are informative and not time-consuming. Moreover, they show that the main methods used to

estimate the protein contents based on the tryptophan, tyrosine,and phenylalanine absorptions are not suitable for such SOM.

There is good agreement between the results of HPLC andelectrophoreses. Despite the use of dissociative buffers in bothtechniques, large molecular size species are observed (�103

kDa).Comparison with Other Calcitic Prisms—Addadi et al (38)

and Albeck et al. (36) have studied the SOM extracted from theouter calcitic prisms of Atrina (Pteriomorpha, Pinnidae). At-rina and Pinna are taxonomically related. Infrared spectra ofAtrina show a main peak at 1653 cm�1, strong bands at 1575and 1417 cm�1, and weak specific sugar bands. The band at1160–1630 cm�1 is assigned to protein amide I but also “pos-sibly to the N-acetyl groups of polysaccharide” (36). Bands at1255 and 1230 cm�1 may be attributed to amide III and/orsulfate (36). However, an acidic part of the matrix shows alarge specific sugar band. Thus, the similarities between theinfrared spectra of P. nobilis and Atrina are high despite thedifferent decalcification processes. The prisms of Atrina alsoshow high contents of acidic amino acids (36). S amino acids(Cys and Met) are low, but the special hydrolysis necessary toavoid destruction of these amino acids was not done by Albecket al. (36).

The Simple Prism Concept—Despite some similar aspects,the microstructures and nanostructures of the prisms of Pinnaand Pinctada are clearly different as previously shown by op-tical microscope studies. Pinna prismatic units are monocrys-talline, whereas those of Pinctada are not. Pinctada SEM ob-servations showed small globular crystals (200–600 nm indiameter) surrounded by a thin granular envelope 3–5-nmthick (17). This size is similar to that of the elongated crystal-lites observed with AFM but is larger than that of the roundedgranules. The ultrastructure of Pinna was not illustrated pre-viously (17). Pinna and Pinctada prisms were also called mega-prisms (39). They are said to be built up of small crystallites

FIG. 10. XANES spectra. A, S amino acids. B, S amino acid with adisulfide bond and CS.

FIG. 11. A, XANES spectra of SOM showing the main S species is SO4in both Pinna and Pinctada and the presence of S amino acids inPinctada. B, in situ XANES spectra in the sinuous organic intrapris-matic membranes of the prisms of Pinctada (see Fig. 2, C–F).

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves 15175

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

(100–300 nm in diameter) and arranged in an irregular patternin Pinctada fucata. No radial intraprismatic membranes wereobserved. Again, the ultrastructure of the prismatic layer ofPinna was not illustrated previously (39).

The combination of analytical methods from simple onessuch as UV and fluorescence to more focused techniques suchas HPLC or XANES allows us to obtain convergent data on theSOM extracted from Pinna and Pinctada calcitic layers. First,UV and fluorescence data show strong differences betweenpure proteins and the SOM, which were studied. The mainfeatures of the infrared spectra are similar, but the main bandin each region is different and indicative of different conforma-tions. These spectra also show that SOM are not pure proteins;sugars and sulfate are present. However, the amount of sugarsis not known and cannot be deduced, because most of the bandsare present in both proteins and sugars.

UV and refractometric HPLC profiles support the conclusionthat the SOM of Pinna and Pinctada are glycoproteins orglycosaminoglycans as well as their different compositions andapparent molecular weights. Both SOM have apparent molec-ular masses higher than 106 Da; thus, only a part of the SOMpenetrates into two-dimensional gels. The different sugar con-tent is also confirmed by the stains. It is often said that theabsence of staining is because of the very acidic makeup of theSOM (40), but both Pinna and Pinctada SOM are very acidic asshown by amino acid compositions (17). The observed differ-ence in silver staining between Pinna and Pinctada is probablybecause of the difference in their sugar contents. It must benoted that Alcian Blue or Acridine Orange stains are not sen-sitive compared with silver staining.

Non-denaturing IEF also shows strong acidity based on theposition of the bands, whereas the high S contents and thedifferences in sugar contents are demonstrated by the stains.Both Pinna and Pinctada SOM contain up to 79% aspartic acid(17, 18), but the amino acid composition is dependent on thespecies. For example, aspartic acid varies from 23 to 76% in twoPinctada species, whereas glutamic acid varies from 3.6 to 18%in two Pinna species (17). XANES spectra confirm that themain sulfur species is sulfate in both SOM, but S amino acidsare also present in Pinctada. Previous microprobe analyseshave shown that these calcitic prisms have high S contents,typically 5650 ppm in Pinna and 3100 ppm in Pinctada (13, 14).Disulfide bonds, if present, are rare as confirmed by liquidchromatography (HPLC) and electrophoresis data. The use ofdissociative denaturant-containing detergent buffers does notresult in any improvement in resolution of the SOMcomponents.2

CONCLUSION

The techniques used do not allow us to identify the glycopro-teins precisely and/or their core proteins or sugar moieties. CSis one of the most widespread sulfated sugars and is frequentlyobserved in extracellular matrices involved in calcification pro-cesses (41–43) and in shell formation (44, 45), but the dataavailable for Pinna and Pinctada are not sufficient to ascertainits presence in these species. The high molecular weights andsugars in Pinna suggest the presence of mucins. Mucins arehigh molecular weight, heavily glycosylated, and sometimessulfated proteins and chromatograph in the void volume ofmost commercially available gels. They do not penetrate wellinto electrophoresis gels. They are stained in situ or in gelswith Alcian Blue. The subunits are joined by disulfide bonds.However, the amino acid contents of mucins are different fromthose of Pinna and Pinctada SOM. Mucins have high threo-

nine, serine (20%), and proline (10%) contents and low asparticacid (4%) and glutamic acid (8%) contents.

Despite some common features, the Pinna and PinctadaSOM are not mucins. SOM extracted from the calcitic prismaticlayer of Pinna is probably composed of a peptide core of repeat-ing units decorated with carbohydrate chains. Pinctada SOM isalso probably composed of a peptide core of repeating units,despite a low glycosylation level and low sulfate content.

Further studies are required to isolate and characterize thecore proteins and the carbohydrate moieties of the SOM ofmollusk shells and the relationships between the extrapallialfluid and the SOM. Hattan et al. (46) have shown that themajor protein of extrapallial fluid of Mytilus is a glycoprotein“that is a homodimer composed of 14.3% carbohydrate.” Dena-turing gel electrophoresis of this protein under reducing ornon-reducing conditions indicates the lack of disulfide bonds.The estimated molecular mass of the subunit is 28,350 Da, andthe pI is 4.43. The major secondary structures are �-helix andthen �-sheet. The Mytilus shell is composed, as are Pinnaand Pinctada, of an inner aragonitic layer and an outer calciticone. However, a direct comparison between the extrapallialfluid and SOM of the calcitic layers remains difficult, becausethe extrapallial fluid is involved in the secretion of all of thelayers of the shells and it is well known that in a single shellthe SOM of the nacreous layer and of the prismatic layer aredifferent.

High S contents, high sugar contents, and sulfate as mainsulfur species are not exclusively known in calcitic minerals.Similar compositions have been described in the SOM ex-tracted from the aragonitic skeletons of Scleractinia (28). Sul-fated mucopolysaccharides are known to be closely associatedwith the mineralization process, and their involvement in thenucleation process of calcitic and aragonitic biominerals in vivohas been shown in various taxa (9, 34, 47–50). Moreover, fromhistochemical and microscopic studies, Wada (47) has shownthat acidic sulfated mucopolysaccharides are not present innon-calcified organic matrices of mollusk shells.

The pioneer results of Wada (47) showing differences in theorganic contents of the calcitic prisms of Pinna attenuata andP. martensii on the one hand and the presence of componentssimilar to CS on the other hand are confirmed. Although theyare present at or before the beginning of the calcification, fewdata are available on the structure and the composition of thethick organic walls or interprismatic sheaths. A detailed studyof the organic matrices of the prismatic layer of Pinna showsthat the interprismatic walls consist of soluble and insolublefractions (31). Nevertheless, the role of each fraction in thebiomineralization process is not explained. Similar data are notavailable for the interprismatic walls of Pinctada.

The simple prism concept is not corroborated by other micro-scopic observations despite a common mineralogy. In manyways, the prisms of Pinna and Pinctada are not identical. TheSOM differ markedly in their structures and composition andin the molecular weights and acidities of their molecular con-stituents. The “similar” features of these prisms are less nu-merous than the “different” features. Both Pinna and Pinctadaare Pteriomorpha bivalves, but they belong to distinct orders.The main microstructural differences (composite structure inPinctada) and the major compositional differences (sugar con-tents) probably reflect fundamental evolutionary trends. Thus,the simple prism concept is not a relevant model for the bi-omineralization processes in the prismatic calcitic layer of mol-lusk shells.

From a methodological point of view, highly focused studieson the protein contents of biominerals are not sufficient toestimate the phylogenetic similarities of taxa on the one hand2 Y. Dauphin, unpublished data.

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves15176

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

and to understand the role of the organic matrices in biomin-eralization processes on the other hand. The next step of thiscomparison is a separation of sugars and proteins followed by aseparation of the constituents of each fraction. From a geolog-ical point of view, the observed distinct characteristics imply adifferent behavior during the fossilization processes despite acommon size and mineralogy.

Acknowledgments—The research carried out at European Synchro-tron Radiation Facility (ESRF) was under contract CH721 and CH948.I thank Dr. J. Susini and Dr. M. Salome (ID 21, ESRF, Grenoble,France) and Pr. Dr. J. Doucet (Laboratoire de l’Utilisation da Rayon-nements Electromagnetiques, Universite Paris XI-Orsay, Orsay,France) for their valuable advice and support. I am grateful to ananonymous reviewer for advice, which greatly enhanced the quality ofthis paper.

REFERENCES

1. Weiner, S., and Hood, L. (1975) Science 190, 987–9892. Weiner, S., and Traub, W. (1984) Philos. Trans. R. Soc. Lond-Biol. Sci. 304,

425–4343. Krampitz, G., Engels, J., and Cazaux, C. (1976) in The Mechanisms of Miner-

alization in the Invertebrates and Plants (Watabe, N., and Wilbur, K. M.,eds) pp. 155–173, University of South Carolina Press, Columbia, SC

4. Samata, T., Sanguansri, P., Cazaux, C., Hamm, M., Engels, J., and Krampitz,G. (1980) in The Mechanisms of Biomineralization in Animals and Plants(Omori, M., and Watabe, N., eds) pp. 37–47, Tokai University Press, Tokyo

5. Samata, T. (1990) The Veliger 33, 190–2016. Nathusius-Konigsborn, W. (1877) Research on the Non-cellular Organisms,

Namely the Crustacean Shields, the Molluscan Shells, and the Eggshells, p.144. Berlin

7. Moynier de Villepoix, R. (1892) J. Physiol. (Paris), 461–518, 582–6748. Wada, K. (1956) Bull. Natl. Pearl Res. Lab. 1, 1–69. Wada, K. (1961) Bull. Natl. Pearl Res. Lab. 7, 703–828

10. Gregoire, C. (1961) Bull. Inst. R. Sci. Nat. Belg. 37, 1–3411. Taylor J. D., Kennedy, W. J., and Hall, A. (1969) Bull. Br. Mus. (Nat. Hist.)

Zool. 3, 1–12512. Wise, S. (1969) SEM Proceedings of the 2nd Annual Symposium, Chicago, IL,

April 1969, pp. 205–21513. Masuda, F., and Hirano, M. (1980) Sci. Rep. Inst. Geosci. Univ. Tsukuba B/1,

163–17714. Cuif, J. P., Dauphin, Y., Flamand, D., Frerotte, B., and Gautret, P. (1986) C. R.

Acad. Sci. (Paris) 303, 251–25615. Marin, F., Muyzer, G., and Dauphin, Y. (1994) C. R. Acad. Sci. (Paris) 318,

1653–165916. Dauphin, Y., and Cuif, J. P. (1999) Ann. Sci. Nat. 2, 73–8517. Nakahara, H., Kakei. M., and Bevelander. G. (1980) Venus Jpn. J. Malacol. 39,

167–17718. Cuif, J. P., Flamand, D., Frerotte, B., Chabin, A., and Raguideau, A. (1987)

C. R. Acad. Sci. (Paris) 304, 475–47819. Watabe, N., and Wada, K. (1956) J. Fac. Fish. Prefect. Univ. Mie. 227–232

20. Boggild, O. B. (1930) D. Kgl. Danske Vidensk. Selsk. Skr. Naturvidensk. ogMathem. 9/2, 231–326

21. Cuif, J. P., Denis, A., and Raguideau, A. (1983) Haliotis 13, 131–14122. Lowenstam, H. A., and Weiner, S. (1989) Biomineralization, Oxford University

Press, New York23. Samata, T., Hayashi, N., Kono, M., Hasegawa, K., Horita, C., and Akera, S.

(1999) FEBS Lett. 462, 225–22924. Fremy, E. (1855) Ann. Chim. (Paris) 43, 47–10725. Cuif, J. P., Dauphin, Y., and Gautret, P. (1988) C. R. Acad. Sci. (Paris) 307,

1943–194826. Cuif, J. P., Dauphin, Y., Gautret, P., and Raguideau, A. (1989) Bull. Soc. Geol.

Fr. 8, 569–57527. Dauphin, Y., and Cuif, J. P. (1997) Electrophoresis 18, 1180–118328. Dauphin, Y. (2001) Int. J. Biol. Macromol. 28, 293–30429. Venyaminov, S. Yu., and Kalnin, N. N. (1990) Biopolymers 30, 1243–125730. Cael, J. J., Winter, W. T., and Arnott, S. (1978) J. Mol. Biol. 125, 21–4231. Dauphin, Y. (2002) Comp. Biochem. Physiol. Part A Physiol. 132, 577–59032. Rose, K., Shadle, S. E., Eidiness, M. K., Kurtz, D. M., Jr., Scott, R. A., Hedman,

B., Hodgson, K. O., and Solomon, E. I. (1998) J. Am. Chem. Soc. 120,10473–10747

33. Prange, A., Arzberger, I., Engemann, C., Modrow, H., Schumann, O., Truper,H. G., Steudel, R., Dahl, C., and Hormes, J. (1999) Biochim. Biophys. Acta1428, 446–454

34. Termine, J. D., Belcourt, A. B., Conn, K. M., and Kleinman, H. K. (1981)J. Biol. Chem. 256, 10403–10408

35. Worms, D., and Weiner, S. (1986) J. Exp. Zool. 237, 11–2036. Albeck, S., Addadi, L., and Weiner, S. (1996) Chem. Eur. J. 2, 278–28437. Aizenberg, J., Lambert, G., Weiner, S., and Addadi, L. (2002) J. Am. Chem.

Soc. 124, 32–3938. Addadi, L., Berman, A., and Weiner, S. (1991) in Mechanisms and Phylogeny

of Mineralization in Biological Systems (Suga, S., and Nakahara, H., eds)pp. 29–33, Springer-Verlag New York Inc., New York

39. Suzuki, S., and Uozumi, S. (1981) J. Fac. Sci. Hokkaido Univ. 1, 7–2040. Killian, C. E., and Wilt, F. H. (1996) J. Biol. Chem. 271, 9150–915941. Weiss, I. M., Kaufmann, S., Mann, K., and Fritz, M. (2000) Biochem. Biophys.

Res. Commun. 267, 17–2142. Tabak, L. A. (1995) Annu. Rev. Physiol. 57, 547–56443. Lechene de la Porte, P., Domingo, N., van Wijland, M., Groen, M., Ostrow,

J. D., and Lafont, H. (1996) J. Hepatol. 25, 339–34844. Prezant, R. S. (1990) in The Bivalvia: Proceedings of a Memorial Symposium

in Honour of Sir Charles Maurice Yonge, Edinburgh 1986 (Morton, B., ed)pp. 83–95, Hong Kong University Press, Hong Kong

45. Marxen, J. C., and Becker, W. (1997) Comp. Biochem. Physiol. 118, 23–3346. Hattan, S. J., Laue, T. M., and Chasteen, N. D. (2001) J. Biol. Chem. 276,

4461–446847. Wada, K. (1964) Bull. Jpn. Soc. Scient. Fish. 30, 993–99848. Crenshaw, M. A., and Ristedt, H. (1976) in The Mechanisms of Mineralization

in the Invertebrates and Plants (Watabe N., and Wilbur, K. M., eds) pp.355–367, University of South Carolina Press, Columbia, SC

49. Wada, K. (1980) in The Mechanisms of Biomineralization in Animals andPlants (Omori, M., and Watabe, N., eds) pp. 79–92, Tokai University Press,Tokyo

50. Fernandez, M. S., Moya, A., Lopez, L., and Arias, J. L. (2001) Matrix Biol. 19,793–803

Soluble Organic Matrices of Prismatic Shell Layers of Two Bivalves 15177

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Yannicke DauphinPteriomorphid Bivalves: PINNA NOBILIS AND PINCTADA MARGARITIFERA

Soluble Organic Matrices of the Calcitic Prismatic Shell Layers of Two

doi: 10.1074/jbc.M204375200 originally published online February 7, 20032003, 278:15168-15177.J. Biol. Chem. 

  10.1074/jbc.M204375200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/278/17/15168.full.html#ref-list-1

This article cites 39 references, 4 of which can be accessed free at

by guest on August 14, 2016

http://ww

w.jbc.org/

Dow

nloaded from