Abstract In zooxanthellate corals, the photosynthetic

fixation of carbon dioxide and the precipitation of

CaCO3 are intimately linked both spatially and tem-

porally making it difficult to study carbon transport

mechanisms involved in each pathway. When studying

Tubastrea aurea, a coral devoid of zooxanthellae, we

can focus on carbon transport mechanisms involved

only in the calcification process. We performed this

study to characterize T. aurea carbonic anhydrase and

to determine its role in the calcification process. We

have shown that inhibition of tissular carbonic anhy-

drase activity affects the calcification rate. We have

measured the activity of this enzyme both in the tissues

and in the organix matrix extracted from the skeleton.

Our results indicate that organic matrix proteins, which

are synthesized by the calcifying tissues, are not only

structural proteins, but they also play a crucial catalytic

role by eliminating the kinetic barrier to interconver-

sion of inorganic carbon at the calcification site. By

immunochemistry we have demonstrated the presence

of a protein both in the tissues and in the organic

matrix, which shares common features with prokary-

otic carbonic anhydrases.

Keywords Carbonic anhydrase � Carbon �Calcification � Coral � Biomineralization �Organic matrix

Abbreviations

CA Carbonic anhydrase

BSA Bovine serum albumin

DIC Dissolved inorganic carbon

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetate

FSW Filtered seawater

PBS Phosphate buffered saline

PAF Paraformaldehyde

PIC Protease inhibitor cocktail

RT Room temperature

SOM Soluble organic matrix

SDS Sodium dodecyl sulphate

TBS Tris buffered saline

DIC Dissolved inorganic carbon

Introduction

Scleractinians (stony corals) are coelenterates that form

aragonitic calcium carbonate (CaCO3) skeletons. They

are classically functionally divided into two groups:

the hermatypic (reef-building) and the ahermatypic

(non-reef-building) corals. The vast majority of the

hermatypic corals are found in shallow, tropical oceans

and characteristically contain within their tissues

large populations of symbiotic dinoflagellates called

Communicated by S.A. Poulet, Roscoff.

S. Tambutte (&) � E. Tambutte � D. Zoccola �N. Caminiti � S. Lotto � A. Moya � D. AllemandCentre Scientifique de Monaco, av. Saint Martin,MC 98 000, Monacoe-mail: stambutte@centrescientifique.mc

J. AdkinsDepartment of Geology and Planetary Sciences, MS100-23,Caltech, 1200 E. California Blvd., Pasadena, CA 91125,USA

D. AllemandUMR 1112 UNSA-INRA Faculte des Sciences,Parc Valrose, B.P.71, 06108 Nice Cedex 2, France

Mar Biol

DOI 10.1007/s00227-006-0452-8

123

RESEARCH ARTICLE

Characterization and role of carbonic anhydrasein the calcification process of the azooxanthellate coralTubastrea aurea

Sylvie Tambutte Æ Eric Tambutte Æ Didier Zoccola Æ Natacha Caminiti ÆSeverine Lotto Æ Aurelie Moya Æ Denis Allemand Æ Jess Adkins

Received: 8 May 2006 / Accepted: 9 August 2006� Springer-Verlag 2006

zooxanthellae. In these zooxanthellate scleractinians,

the photosynthetic fixation of carbon dioxide (CO2)

and precipitation of CaCO3 are intimately linked both

at spatial (cell to ecosystem) and temporal (day–night)

scales rendering it difficult to study the carbon transport

mechanisms involved in each pathway. On the other

hand, the vast majority of ahermatypic corals are de-

void of zooxanthellae. Thus when studying these corals,

it is possible to focus on carbon transport mechanisms

involved only in calcification processes.

In corals, skeleton formation is a process of

‘‘extracellular biologically-controlled biomineraliza-

tion’’ and as such involves a mineral fraction and an

organic matrix. The means by which corals may

influence CaCO3 precipitation include (1) control of

the levels of inhibitors, promoters, and regulators of

calcification by the means of a set of macromolecules

(called the organic matrix) surrounding the crystal or

included within the mineral and (2) availability of

substrates. Since coral skeleton formation results from

the delivery of calcium and inorganic carbon to the

site of calcification, these two substrates are crucial to

study. Recently, most of the research involving coral

calcification has focused either upon the structure and

composition of organic matrices of skeletons (Gautret

et al. 1997, 2000; Cuif et al. 1999, 2003; Dauphin

2001) or on the uptake and mechanisms of deposition

of calcium ions (Wright and Marshall 1991; Tambutte

et al. 1995, 1996). However, for invertebrate miner-

alization, carbonate ions are as important as calcium

ions. Pearse (1970) established that skeletal carbonate

can originate from two different carbon sources: sol-

uble carbonates from sea water or CO2 produced by

animal metabolism. Furla et al. (2000) demonstrated

that in the zooxanthellate coral Stylophora pistillata,

the major source of DIC for coral calcification is

metabolic CO2 and not inorganic carbon originating

from seawater. Similar results were obtained in the

non-zooxanthellate octocoral Leptogorgia virgulata

and Corallium rubrum respectively by Lucas and

Knapp (1997) and Allemand and Grillo (1992). In

addition, these last authors have shown that DIC

supply is rate-limiting for calcification. However,

these conclusions can not be generalized to all corals

since Adkins et al. (2003) demonstrated that there is

little or no metabolic CO2 in the skeleton of the

deep-sea non-zooxanthellate coral, Desmophyllum

cristagalli.

Carbonic anhydrases are ubiquitous enzymes

known to act as catalysts for the interconversion

between CO2 and HCO3–. Since the limiting step in

the conversion from CO2 to carbonate ion is the

hydration step, CA can play an important role when

calcification is carbon limited. In avians, CA facili-

tates eggshell formation (Nys and de Laage 1984) and

in fishes, CA is supposed to play an important role in

otolith formation (Payan et al. 1997; Tohse and

Mugiya 2001; Tohse et al. 2004). In the case of

invertebrates, this enzyme has been found to play a

role in the calcification of calcareous sponges (Jones

and Ledger 1986), scleractinian corals (Goreau 1959;

Isa and Yamazato 1984; Marshall 1996, Furla et al.

2000; Al-Horani et al. 2003), octocorallians (Kingsley

and Watabe 1987; Allemand and Grillo 1992; Lucas

and Knapp 1996, 1997; Rahman et al. 2005, 2006),

molluscs and echinoderms (Miyamoto et al. 1996;

Mitsunaga et al. 1986). Carbonic anhydrase has been

described in many tissues but its presence in extra-

cellular calcified structures suggests that this enzyme

could also play an important role during the precipi-

tation step of the mineral. The aim of this study was

to characterize T. aurea carbonic anhydrase and

determine its role in the calcification process.

Materials and methods

Biological material

Parent coral colonies of Tubastrea aurea (Cœlentera-

ta:Anthozoa:Scleractinia), indigenous to the Indo-Pa-

cific, and sea anemones Aiptasia pulchella were

maintained at the Centre Scientifique de Monaco in

the following conditions: semi-open circuit, Mediter-

ranean sea water heated to 26 ± 0.2�C, and fed with

Artemia nauplii twice a week. T. aurea was maintained

in low light conditions at a constant irradiance

of 15 lmol photons m–2 s–1 on a 12 h/12 h light/dark

cycle, and A. pulchella was maintained in the same

conditions but with a constant irradiance of 250 lmol

photons m–2 s–1. For calcification rate experiments,

coral colonies were cut with a bone cutter in order to

obtain fragments of three to four polyps called nub-

bins. The sectioned skeleton was coated with epoxy

resin so that only the tissues were in contact with

seawater. Nubbins were used for experiments after a

period of 4–5 weeks, when new tissues entirely covered

the junction between the resin and the skeleton.

Cleaning was performed daily.

Preparation of tissues for carbonic anhydrase

activity assay

Six to seven polyps were cut from parent colonies, put

on ice and homogenized with a mortar in about 3 ml

cold veronal buffer prepared according to Weis et al.

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123

(1989): 25 mM veronal containing 5 mM ethylene-

diaminetetraacetate (EDTA), 5 mM dithiothreitol

(DTT), 10 mM MgSO4 with pH adjusted to 8.2. Pro-

tease inhibitor cocktail (PIC, SIGMA) 0.1% was then

added. The mixture was sonicated for 1 min and then

centrifuged at 765g for 20 min at 4�C. The supernatant

was centrifuged again in the same conditions and then

aliquoted in Eppendorf tubes for storage at –80�C.

Preparation of organic matrix for carbonic

anhydrase activity assay

Polyps were cleaned by removing soft tissues with 2N

NaOH for 2 h at 70�C. The skeletons were rinsed with

ultrapure water, dried at 60�C overnight and ground to

a fine powder with a mortar. The powder was demin-

eralized for one night at 4�C in 0.5 M EDTA con-

taining 0.1% PIC and 5 mM phenanthrolin. After

complete dissolution of aragonite, centrifugation

(10 min, 10,000g, 4�C) allowed soluble and insoluble

matrices to be separated. To desalt soluble compo-

nents, the supernatant containing the soluble organic

matrix (SOM) was filtered and concentrated using

Centricon� (Amicon, cut-off 5 kDa) according to the

manufacturer’s instructions. The retentate was ali-

quoted and stored at –80�C.

In vitro assay for carbonic anhydrase activity

The in vitro assay for CA activity is described in detail

by Weis et al. (1989). Briefly CA activity in crude ho-

mogenates was measured by the decrease of pH

resulting from the hydration of CO2 to HCO3– and H+

after the addition of substrate. All experiments were

performed at 4�C. CO2–saturated distilled H2O served

as a substrate and was prepared by passing gaseous

CO2 through an airstone for 30 min (pH 3.5). To run

the assay, 5 ml veronal buffer (pH 8.2) were trans-

ferred to a small beaker and 1 ml of homogenate

diluted in veronal buffer was added to obtain quanti-

ties of proteins ranging from 0 to 12 mg for tissues and

0–16 lg for organic matrix. The mixture was constantly

stirred with a magnetically driven stirring bar. Four ml

of substrate was then added rapidly and the decrease in

pH was recorded by a Ag/AgCl pH probe immersed

in the mixture and connected to a Metler DL70 pH

meter fitted with a chart recorder. As a control for

the non-catalyzed reaction, the same experiment was

performed without homogenate.

Carbonic anhydrase activity was calculated as

(t0 – t)/t, where t0 is the time needed for the non-

catalyzed reaction and t is the time for the catalyzed

reaction to obtain a pH decrease from 8 to 7.5. Units of

enzyme activity (EU) were normalized to the weight of

soluble proteins.

Inhibition of carbonic anhydrase activity

To test the effect of the inhibitor ethoxyzolamide on

CA activity, the assay was performed as described

above, but 0–10 lM ethoxyzolamide was added to the

veronal buffer before addition of tissue. Results are

expressed as percent inhibition calculated from 100 –

[(CA activity in presence of inhibitor/CA activity

in absence of inhibitor) · 100)]. IC50 represents the

concentration of inhibitor, which inhibits half of the

enzyme activity measured in the absence of inhibitor.

Effect of inhibition of CA activity on calcification

rate

Measurement of calcification rate was made according

to the method of Tambutte et al. (1995) adapted for

higher volumes. Measurements were made at equiva-

lent times of day in order to avoid possible variation

caused by endogenous circadian rhythms (Buddemeier

and Kinzie 1976). Nubbins grown on epoxy resin were

incubated for 2 h 15 min in 60 ml beakers containing

approximatively 800 kBq of 45Ca (as 45CaCl2, NEZ013,

Perkin Elmer) dissolved in filtered seawater. For

inhibition experiments, 10 lM ethoxyzolamide was

added in the incubation medium. Water motion was

provided during each incubation by small stirring bars

in order to reduce as much as possible diffusion limi-

tation by boundary layers. Exposure to air was limited

to less than 5 s during transfer to the incubation

beakers and incubations were made under low light

conditions.

At the end of the labelling period, each nubbin was

immersed for 20 s in a beaker containing 1 l FSW.

Labelled nubbins were then incubated in a beaker

containing 150 ml FSW for 180 min to monitor 45Ca

efflux into the rinse medium. Water motion was pro-

vided in the efflux medium by stirring bars. Upon

completion of the efflux, nubbins were dissolved

completely over a period of 20 min in approximately

5 ml of 1 N NaOH at 90�C. Each skeleton was then

rinsed six times in 5 ml of distilled water, dried and

dissolved in 10 ml of 6 N HCl overnight (‘‘HCl-soluble

fraction’’).

Radioactive samples were added to 4 ml Ultima

Gold (Packard) and emissions were measured using a

liquid scintillation analyzer (Tricarb, 2100TR, Pack-

ard).

Mar Biol

123

For the efflux, results are expressed as dpm mg–1

protein. For the calcification rate, results are expressed

as nmol Ca2+ h–1 mg–1 protein in tissues or nmol

Ca2+ h 1 g–1 skeleton and represent means ± S.D. for

at least three measurements. Calculation of the half-

time for calcium washout (T1/2) and its corresponding

rate constant as well as calculation of the size of the

coelenteric pool were made according to Tambutte

et al. (1995). In the presence of ethoxyzolamide, results

are expressed as percentage inhibition of calcification

rate.

A control experiment was performed with killed

nubbins (paraformaldehyde) in order to determine non

specific binding of 45Ca.

Extraction of proteins for immunoblotting

• Proteins from tissues: Six polyps of T. aurea were

cut from a colony with a bone cutter and rinsed in

FSW. They were then homogenized with a mortar

maintained on ice, in about 8 ml of extraction

buffer (50 mM Tris, 100 mM NaCl, 5 mM EDTA,

1% Triton X100, 0.1% PIC, and 5 mM phenanthr-

olin). The mixture was twice centrifuged at 765g for

10 min at 4�C to eliminate skeletal debris. The

supernatant was maintained on ice for 20 min while

vortexing every 5 min, the time necessary for the

buffer to extract proteins. The supernatant was

aliquoted in Eppendorf tubes for storage at –80�C

before experiments. Proteins were also extracted

from the sea anemone A. pulchella with the same

protocol except that they were directly homoge-

nized in the extraction buffer and sonicated.

• Proteins from organic matrix: The protocol was the

same as the preparation of organic matrix for the

carbonic anhydrase activity assay (see paragraph

above).

Dot blot

The activity of the antibody was examined by a dot-

blot assay on proteins extracted either from whole

tissues (40 lg of proteins) or organic matrix (12 lg of

proteins). Experiments were performed at room tem-

perature.

Briefly, the samples were deposited on nitrocellulose

membranes which were saturated with 1% BSA for 1 h

in TBS (140 mM NaCl, 5 mM Tris, pH 7.4) and la-

belled for 1 h with primary antibodies. The primary

antibodies were either (1) anti phycoerythrin (Ab-

Cam), 1:20,000 dilution or (2) rabbit anti-human

erythrocyte carbonic anhydrase II antibody (Rockland

immunochemicals), 1:10,000 dilution or (3) rabbit anti

N-terminal b-carbonic anhydrase from Synecchococcus

sp (generous gift from Mak Saito and Francois Morel),

1:25,000 dilution, in TBS-BSA 1%). Membranes were

then rinsed and incubated for 1 h with secondary

antibodies (horseradish peroxidase-linked anti-rabbit

IgG, Sigma, 1:2,000 dilution in TBS-BSA 1%).

Immunoreactive dots were then revealed with ECL kit

(GE Healthcare). Controls were made with the pre-

immune serum as the primary antibody.

Electrophoresis, protein transfer and Western blot

Proteins extracted either from whole tissues (100 lg

of proteins) or just the organic matrix (20 lg of pro-

teins) were homogenized in Laemmli sample buffer

(Laemmli 1970). Samples were resolved in SDS–

PAGE (12% acrylamide for resolving gel, 4% acryl-

amide for stacking gel) using a Mini Protean II

apparatus (BIORAD). Proteins were then electro-

phoretically transfered from unstained gels onto

PVDF membranes using a transfer apparatus (Mini

Transblot Cell, BIORAD). After transfer, membranes

were saturated with 5% skimmed milk in TBS con-

taining 0.1% Tween and labelled for 1 h with primary

antibodies either (1) anti phycoerythrin 1:20,000

dilution, or (2) anti-human erythrocyte carbonic an-

hydrase II antibody, 1:10,000 dilution, or (3) rabbit

anti-b carbonic anhydrase from Synecchococcus sp.,

1:10,000 dilution, in TBS containing 1% skimmed

milk and 0.1% Tween� 20). Membranes were then

rinsed and incubated for 1 h with secondary anti-

bodies (horseradish peroxidase-linked anti-rabbit IgG,

Sigma, 1:2,500 dilution in TBS containing 1% skim-

med milk and 0.1% Tween� 20). Immunoreactive

dots were then revealed with an ECL kit (GE

Healthcare). Controls were made with the preimmune

serum as the primary antibody.

Preparation of samples for immunolocalization

• Demineralized samples: One polyp was fixed in 3%

paraformaldehyde in S22 buffer (NaCl 450 mM,

KCl 10 mM, MgCl2 58 mM, CaCl2 10 mM, Hepes

100 mM, pH 7.8) at 4�C overnight and then

decalcified using 0.5 M EDTA in Ca-free S22 with

3% PAF at 4�C. It was then dehydrated in an

ethanol series, cleared with xylene and embedded

in Paraplast. Cross sections (7 lm thick) were cut

and mounted on silane-coated glass slides.

• Mineralized samples: One polyp including skele-

ton was fixed in 3% paraformaldehyde in S22

Mar Biol

123

buffer (NaCl 450 mM, KCl 10 mM, MgCl258 mM, CaCl2 10 mM, Hepes 100 mM, pH 7.8)

at 4�C overnight. It was then dehydrated in

ethanol and embedded in LR White resin. Sec-

tions were cut with a low speed saw (Buehler,

Isomet) in thick slices (about 1 mm), etched with

EDTA 1% for 1 h to expose antigenic epitopes

and rinsed in ultrapure water.

Immunolocalization of carbonic anhydrase

Deparaffinized sections of tissues or samples of skele-

ton prepared as described above were saturated with

5% BSA in 0.05 M PBS, pH 7.4, containing 0.2%,

teleostean gelatin, 0.2% Triton X100. The samples

were then incubated with primary antibodies from

rabbit anti-b carbonic anhydrase 1:1,000 dilution in

BSA-saturated PBS solution (PBS 0.05 M, pH 7.4,

containing 0.2%, teleostean gelatin, 0.2% Triton X100,

5% BSA), 1 h at RT and overnight at 4�C in moist

chamber. After rinsing in BSA-saturated PBS solution,

they were incubated with biotinylated anti-rabbit

antibodies (GE Healthcare 1:250 dilution, 1 h at RT)

as secondary antibodies. They were finally incubated

for 20 min with streptavidin-Alexa Fluor 568 (Molec-

ular probes, 1:50 dilution) and DAPI (2 lg ml–1, SIG-

MA). Controls were routinely performed with the

rabbit preimmune serum as the primary antibody.

Samples were embedded in Pro-Long antifade medium

(Molecular probes) and observed with a confocal laser

scanning microscope (Leica, TCS4D) at 568 nm exci-

tation, 600 nm emission.

Histology

Cross sections of demineralized samples or thick slices

of mineralized samples (see paragraphs above for

preparation) were stained with hemalun, eosin, and

acetified anilin blue solutions.

Media and chemicals

Unless otherwise specified, all chemicals were obtained

from Sigma or Biorad and were of analytical grade.

FSW was obtained by filtering seawater on 0.22 lm

Millipore membranes.

The carbonic anhydrase inhibitor ethoxyzolamide

was dissolved in DMSO to a concentration of 60 mM

and buffered with 1 M Tris to pH 8.2.

Protein concentration was measured in a microplate

using the BCA Protein Assay Kit (Uptima�UP40840A). BSA was used as a standard.

Statistical analysis of the data

The effect of the carbonic anhydrase inhibitor, eth-

oxyzolamide, on calcification rate was tested using a t

test (software Jump 5.1, SAS Institute, Cary, USA).

Results are considered statistically significant when

P < 0.05.

Results

The approach we have taken towards the long-range

goal of understanding the mechanisms of biomineral-

ization in corals has been to characterize Tubastrea

aurea carbonic anhydrase and then to determine its

role in the calcification process. We report results of

the measurement of enzyme activity in tissues and or-

ganic matrix and the effect of inhibition of carbonic

anhydrase on the calcification rate. We have revealed

by Western blotting and immunohistochemistry the

presence of a protein both in the tissues and in the

organic matrix, which reacts with an antiserum against

prokaryotic carbonic anhydrases.

Carbonic anhydrase activity

Measurements performed with varying concentrations

of tissues or organic matrix showed that, in both cases,

the activity increases as a linear function of protein

quantity (Fig. 1a, b). When normalized to mg of pro-

teins, a mean value of 0.084 units of enzyme activity

was obtained for tissues and a mean value of 61.6 units

of enzyme activity was obtained for organic matrix.

This difference in the activity value may be due to a

standardization artefact (see Discussion). Dose–re-

sponse experiments were performed with the homo-

genates of tissues (Fig. 2a) or the organic matrix

(Fig. 2b) in the presence of the carbonic anhydrase

inhibitor, ethoxyzolamide. The IC50 was 200 times

higher for the organic matrix (600 nM) than for the

tissues (3 nM). Organic matrix boiled at 100�C for

10 min did not show any carbonic anhydrase activity.

Effect of inhibition of carbonic anhydrase activity

on the calcification rate

In order to determine if carbonic anhydrase was

involved in the calcification process, we measured

the uptake and deposition of calcium in the absence

and in the presence of the carbonic anhydrase inhibitor

ethoxyzolamide. We first determined the efflux time

corresponding to the emptying of 45Ca from the coel-

enteric compartment (Tambutte et al. 1995). Figure 3

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123

shows the kinetics of 45Ca efflux from nubbins loaded

during 135 min in labelled seawater. 45Ca released

from nubbins displays a saturation curve with a plateau

reached within 100 min of efflux. Semi-logarithmic

treatment of the results (inset of Fig. 3) indicates a

T1/2 (half-time of exchange) for calcium washout of

25 min corresponding to a rate constant of 0.028 min–1.

The volume of the coelenteric compartment calculated

at equilibrium gives a value of 12.71 ± 3.6 ll mg–1

protein.

Following this experiment, an efflux time of 180 min

was chosen to completely rinse the coelenteric cavity.

We determined that incorporation of 45Ca in killed

samples was less than 1% and thus could be considered

as negligible. We measured the calcification rate

(deposition of calcium in the skeleton) in the absence

and in the presence of ethoxyzolamide (Fig. 4). The

results show that the incorporation of Ca2+ in the HCl-

soluble pool corresponding to the skeleton has a value of

5.88 ± 0.81 nmol Ca2+ h–1 mg–1 protein or if expressed

per g of skeleton, 0.43 ± 0.09 lmol Ca2+ h–1 g–1skele-

ton. Ethoxyzolamide significantly affected this uptake

(t test, P = 0.004).

1.2

1.0

0.8

0.6

0.4

0.2

0.0

CA

act

ivit

y (E

U)

1614121086420

Quantity of proteins (mg 10-3)

1.2

1.0

0.8

0.6

0.4

0.2

0.0

CA

act

ivit

y (E

U)

1614121086420

Quantity of proteins (mg)

A

B

Fig. 1 Carbonic anhydrase activity in tissues (a) and organicmatrix (b) of T. aurea expressed as a function of the quantity ofproteins in the samples. Each point represents the mean of threevalues

100

80

60

40

20

0Per

cen

tag

e o

f in

hib

itio

n

100x10-3806040200

Concentration of EZ (µM)

A

100

80

60

40

20

0Per

cen

tag

e o

f in

hib

itio

n

1086420

Concentration of EZ (µM)

B

Fig. 2 Inhibition of carbonic anhydrase activity of T. aurea inthe presence of ethoxyzolamide. a Tissues. b Organic matrix.Each point represents the mean of three values

1.6x106

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

dp

m.m

g p

rote

in-1

200150100500Time (min)

15.0

14.0

13.0

12.0

Ln

(Q

e-Q

)

100806040200Time (min)

Fig. 3 Kinetics of 45Ca efflux from T. aurea nubbins loaded for2 h in labelled seawater. Data are expressed in dpm (disintegra-tions per minute) and normalized per g of dried skeleton. Eachpoint represents the mean of three values. Inset: Ln(Qe – Q) as afunction of time. Qe represents the equilibrium value and Q thevalue for each time

Mar Biol

123

Immunochemistry

Before using the antibodies for immunolocalization,

we tested their reactivity by immunochemistry. Dot

blots were performed with the proteins/enzymes in

their non denaturated form whereas Western blots

were performed with the proteins/enzymes in their

denaturated form (due to the presence of SDS and b-

mercapto-ethanol during electrophoresis).

For T. aurea, the antibody against human erythro-

cyte carbonic anhydrase II did not give any positive

labelling in either the dot blot (results not shown) or

in the Western blot (Fig. 5a). The sea anemone con-

trol, on the other hand, gave a positive labelling with

a band at a molecular weight of 31 kDa (Fig. 5a). As

shown in Fig. 5b, the antibody against cyanobacterial

b-CA gave a positive result on dot-blots of both the

tissues and the organic matrix, while the control with

preimmune serum gave no response (Fig. 5c). Wes-

tern blots revealed a 35-kDa band for the tissues and

a band of higher apparent molecular mass (37-kDa)

for the organic matrix (respectively Fig. 5d, e, bCA

lanes). The control using a preimmune serum gave no

response (Figs. 5d, e, lanes P). Since Lesser et al.

(2004) have demonstrated the presence of cyanobac-

teria within coral animal cells, we tested if the label-

ling observed with the antibody against cyanobacterial

b-CA was due to cyanobacteria themselves. We used

anti-phycoerythrin, the antibody used by Lesser et al.

(2004), and did not obtain any positive response, thus

indicating that cyanobacteria were not present in the

coral T. aurea (results not shown). Therefore, the

antibody raised against cyanobacterial b-CA will be

used for immunolocalization of CA in the rest of this

paper.

Immunolocalization

Since no histological data were available in the litera-

ture for T. aurea, we first studied the organization of the

tissues around the skeleton. Figure 6a shows the living

polyps we used to prepare histological sections without

or with a skeleton (Fig. 6b–d, respectively). In longi-

tudinal sections, it appears that the skeleton has a very

winding architecture. Immunolabelling of tissues shows

Fig. 4 Calcification rate of T. aurea nubbins (HCl pool,skeleton) in the absence and in the presence of inhibitors ofCA activity (ethoxyzolamide). Each result represents the meanof three values

Fig. 5 Immunochemistry with anti human erythrocyte carbonicanhydrase II (a) and anti prokaryotic ß-carbonic anhydraseantibody (b–e). a Western blot on tissues of Aiptasia pulchella(lane Aipt.) and Tubastrea aurea (lane Tub.). b Dot-blot on T.aurea tissues (T, 40 lg of proteins) or organic matrix (OM, 12 lgof proteins) on nitrocellulose membrane incubated with the antiprokaryotic ß-carbonic anhydrase antibody (dilution: 1/25,000).c Dot-blot on T. aurea tissues (T, 40 lg of proteins) or organicmatrix (OM, 12 lg of proteins) on nitrocellulose membraneincubated with preimmune serum (dilution: 1/25,000). d ande lanes P: Western blots respectively on T. aurea tissues (100 lg)and organic matrix (20 lg) with preimmune serum (dilution 1/10,000). d and e lanes ß-CA: Western blot respectively on T.aurea tissues (100 lg) and organic matrix (20 lg) with the antiprokaryotic ß-carbonic anhydrase antibody (dilution 1/10,000).OM Organic matrix, T tissues, ßCA anti-prokaryotic ß-carbonicanhydrase antibody, P preimmune serum, Aip Aiptasia pulchella,Tub Tubastrea aurea

Mar Biol

123

that the antibody binds to the tissues facing the skeleton

(Fig. 7a). Preimmune serum was used as a control to

evaluate non-specific immunoreactivity (Fig. 7b), the

faint fluorescence observed was due to the autofluo-

rescence of the tissues (the same faint fluorescence is

observed in control experiments with no preimmune

and no antibody, results not shown). At higher magni-

fication (Fig. 7c, d) it appears that labelling of the tis-

sues is localized in only one layer of cells. When

performed on the skeleton, the labelling appears as a

reticulate network on the envelopes surrounding the

fibres (Fig. 7e). Figure 7f shows that the preimmune

serum gave no background signal on the skeleton.

Discussion

In the present work, we investigated carbonic anhydrase

from the tissues and the skeletal fraction of

T. aurea by (1) measuring the enzyme activity, (2)

determining its involvement in biomineralization, (3)

determining some of its biochemical properties.

Measurement of carbonic anhydrase activity

While it appears that cellular carbonic anhydrase plays a

key role in the availability of carbon involved in differ-

ent physiological processes, there are few studies deal-

ing with the presence and the role of extracellular CA in

the calcium carbonate deposition process. In calcified

skeletal structures of invertebrates, where calcium car-

bonate is the major component, a set of molecules

grouped under the term of ‘‘organic matrix’’ are always

present (Weiner 1984; Wheeler and Sikes 1984;

Constantz and Weiner 1988; Falini et al. 1996). The

roles of these molecules in the calcification process

are numerous: initiation/inhibition of crystal growth,

crystal morphology, and calcium binding (for review

see Wheeler et Sikes 1984; Watanabe et al. 2003).

Miyamoto et al. (1996) were the first to discover a

carbonic anhydrase domain within nacrein, a soluble

organic matrix protein of the nacreous layer in the

mollusc Pinctada fucata. Since then, a cDNA that

encodes a shell matrix protein composed of carbonic

anhydrase-like domains has been cloned in the oyster

Pinctada maxima by Kono et al. (2000). Watanabe et al.

(2003) have also found an internal sequence in T. aurea

that exhibits similarity to a part of the carbonic anhy-

drase sequences. CA activity was also recently found in

various biominerals (Borelli et al. 2003; Rahman 2005,

2006) suggesting a widespread distribution of CA in

calcium carbonate biominerals. In the present work we

measured carbonic anhydrase activity in the organic

matrix of T. aurea. The value of this activity is higher for

organic matrix than for tissues, which can be due either

to (1) a truly higher activity in the organic matrix, (2) a

different CA between tissues and the organic matrix, (3)

an artefact with standardization methods related to

protein assays. Indeed, invertebrate organic matrix

proteins have special biochemical features which render

their characterization difficult, for example when per-

forming staining after electrophoresis (Gotliv et al.

2003). A similar reason could explain the problems

encountered when using protein assays on the organic

matrix that probably underestimate the protein content

(unpublished results). Nevertheless the important point

to consider is that a carbonic anhydrase is present in the

organic matrix and that this enzyme possesses an

activity. Carbonic anhydrase activity in the organic

matrix is inhibited by ethoxylamide and the inhibition

constant IC50 in this matrix is 200 times higher than for

tissues. It is important to note the high resistance of the

organic matrix-linked CA, because its activity is pre-

served after the long demineralization and purification

steps necessary to obtain organic matrix itself.

Determination of CA involvement

in biomineralization

We looked at the role of CA in biomineralization by

using a pharmacological approach. We first measured

the calcification rate in control conditions and then tes-

ted the effect of CA inhibitors. We determined that the

value of calcium deposition in the skeleton is similar (i.e.

0.43 ± 0.09 lmol Ca2+ h–1 g–1 skeleton) to the value

obtained by Marshall (1996) on the same species (i.e.

0.48 ± 0.03 lmol Ca2+ h–1 g–1 skeleton). The volume of

the coelenteric cavity (i.e. 12.71 ± 3.6 ll mg–1 protein) is

comparable to the value obtained for the scleractinian

coral S. pistillata (i.e. 7.3 ± 1.2 ll mg–1 protein, Tambutte

et al. 1995). Our results on the inhibition of calcium

deposition into the skeleton in the presence of ethoxy-

zolamide indicate that CA is involved in the calcification

process. This type of inhibition of the calcification rate

was also observed in hermatypic corals (Goreau 1959;

Tambutte et al. 1996; Furla et al. 2000), sea urchin

spines (Heatfield 1970), barnacle shells (Yule et al. 1982),

molluscans (Wilbur and Jodrey 1955), crustaceans

(Roer 1980) and the red coral Corallium rubrum

(Allemand et al. 1992). Using the same kind of approach

with 45Ca, Kingsley and Watabe (1987) obtained the

opposite results in the gorgonian Leptogorgia virgulata

with an increase of Ca uptake in the spicules and the

axes in the presence of carbonic anhydrase inhibitors.

Nevertheless, while the mechanism of CA action seems

to differ depending on the species, in all cases, the results

show that CA is involved in the calcification process

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123

Depending on the source of carbon used at the site

of calcification, two hypothesis can be proposed:

1. If seawater HCO3– is the source of DIC, CA may

catalyze its conversion to CO2 to buffer the acidity

produced by the conversion of HCO3– into CO3

2–, as

already suggested by Sikes et al. (1980) for cocco-

lithophorids, following the equations:

HCO3- CO3

2- + H+

H+ + HCO3- CA CO2 + H2O

_________________________2 HCO3

- CO32- + CO2 + H2O

2. If intracellular CO2 is the form of DIC used at the

site of calcification, then the extracellular organic

matrix-linked CA may help in converting this CO2,

which diffuses from the tissue to the skeletogenic

fluid due to the probable high pH of this calcifying

region (Furla et al. 2000; Al-Horani et al. 2003),

into HCO3–, following the equations:

CO2 + H2OCA H+ + HCO3

-

HCO3- CO3

2- + H+

_________________________CO2 + H2O 2H+ + CO3

2-

The two H+ produced by these sets of reactions may

then be removed from the site of calcification by the

Ca2+-ATPase present within the calicoblastic epithe-

lium (Zoccola et al. 2004) which catalyzes the exchange

2H+/Ca2+.

Determination of some CA properties

Since we have shown that CA is present in both the

tissues and the organic matrix and we demonstrated

that this enzyme plays a role in the calcification process,

we tried to constrain some of its biochemical properties

and its localization both in the tissues and the organic

matrix. Five groups of cellular CA are described in the

literature (Hewett-Emmett and Tashian 1996; Cox

et al. 2000; Lane et al. 2000, 2005) (1) a-CA, mostly

found in eukaryotes, (2) b-CA, characteristic of eu-

bacteria and plants (3) c-CA, characteristic of archae-

bacteria (4) d-CA and e-CA described in the marine

diatom Thalassiosira weisflogii. The enzymes of the

type alpha, beta, and gamma use zinc as cofactor

whereas delta-CA can switch between zinc, cadmium

and cobalt, and epsilon-CA uses cadmium. Weis and

Reynolds (1999) have shown, by Western blotting with

an antibody raised against human CA, the presence of

an a-carbonic anhydrase in the tissues of a sea anem-

Fig. 6 Structure of T. aureapolyps. a Fresh polyps.b Longitudinal section of ademineralized polyp showingthe tissues. c Longitudinalsection of a nondemineralized polyp.d Higher magnification ofsection C showing tissuesabove the skeleton. Coecoenosarc (tissue betweenpolyps), Mo mouth, Po polyp,Sk skeleton, SW seawater

Mar Biol

123

one. We found the same result in the present study

using the sea anemone A. pulchella. Watanabe et al

(2003) found in T. aurea an internal sequence of a

matrix protein that exhibits sequence similarity with a-

CA sequences. However, in the present study, in T.

aurea, we could not detect CA using an immunological

approach with an anti a-CA antibody. Thus, if an a-CA

is present in T. aurea, it possesses epitopes in the cat-

alytic site that are not recognized by the antibody

against human a-CA. This result could be due to a

difference in structure between mammal a-CA and

coral a-CA. On the other hand, Western blotting with

an antibody raised against a carbonic anhydrase from

Synecchococcus sp. shows that there is a protein that

immunoreacts with this antibody raised against a

prokaryotic CA in both the tissues and in the organic

matrix. Since no labelling was obtained with the anti-

body against phycoerythrin, a cyanobacterial marker,

we can suggest that the labelling observed with the

antibody against the prokaryotic CA is specific and not

due to the presence of cyanobacteria in coral tissues.

The presence of a protein, which shares properties

with enzymes found in prokaryotes appears surpris-

ing. However, prokaryotic-like proteins have already

Fig. 7 Immunolocalization ofprokaryotic ß-carbonicanhydrase respectively in thetissues (a–d) and in theskeleton (e and f) of T. aurea.a, e In orange: carbonicanhydrase antibody coupledto Alexafluor 568. b Controlwith preimmune serum. c, dIn orange: carbonic anhydraseantibody coupled toAlexafluor 568 merged withDAPI staining showing thenuclei in blue. f Control withpreimmune serum showing nosignal in the skeleton. SKSkeleton

Mar Biol

123

been described in Cnidarians. For example, Richier

et al. (2003) found an extra-mitochondrial, monomeric

Mn-superoxide dismutase and a Fe-superoxide dismu-

tase, both enzymes characteristic of prokaryotes, within

the tissues of a sea anemone. More recently, by study-

ing 26,845 ESTs from a coral and a sea anemone,

Technau et al. (2005) found that about 1.3–2.7% of

cnidarian proteins only matched with non-metazoan

sequences (i.e. fungi, prokaryotes, plants, and protists),

many matching only with bacterial sequences. Among

these bacterial sequences, they identified the bacterial

universal stress protein (UspA). To explain this result,

these authors suggested either a conservation of ancient

genes within the genomes of basal metazoans or lateral

gene transfer. It is noteworthy that such observations

are not only limited to cnidarians since in tunicates it

has also recently been suggested that enzymes involved

in cellulose biosynthesis are likely acquired by hori-

zontal transfer from bacteria (Sasakura et al. 2005).

The protein characterized in our Western-blots by

its immunoreactivity with the prokaryotic CA antibody

has an apparent molecular mass in the tissues of

35 kDa, which is smaller than the one found in the

organic matrix (37 kDa). This difference can be ac-

counted for by oligosaccharide chains (Waheed et al.

1992; Wilson et al. 2000) since, in corals, matrices are

highly glycosylated (Dauphin 2001). When chemical

fluorochrome staining or immunolocalization was per-

formed with this antibody on the skeleton, labelling

displayed a pattern typical of the organic matrix

(Gautret et al. 2000; Puverel et al. 2005). The existence

of a protein positively labelled by the same antibody

both in the tissues and in the organic matrix suggests

that high homologies exist between these two proteins,

the latter could be the secreted form of the former. In

this case, however, since we have determined that

carbonic anhydrases react differently to inhibitors, it is

probable that these two enzymes are different iso-

forms. Another possibility is that the pharmacologic

differences between these two isoforms only result

from a modification, before secretion of the protein, by

glycosylation. Further characterization is needed to

solve this point. Furthermore, the differences in

molecular weight observed in Western-blot confirms

that, as in zooxanthellate corals (Puverel et al. 2005),

organic matrix proteins of azooxanthellate corals do

not result from the trapping of the whole soft tissues as

suggested by Constantz (1986). By immunohisto-

chemistry, we showed that only the tissues facing the

skeleton (i.e. the calcifying tissues) are labeled with the

antibody raised against prokaryotic CA, suggesting

that this protein is involved in the biomineralization

process.

Conclusions

Our work was performed on the azooxanthellate coral

T. aurea, allowing us to study the transport of carbon

used for calcification while eliminating the complica-

tions due to photosynthesis. We demonstrated the

presence of an active carbonic anhydrase both in the

tissues and in the organic matrix with a direct role of

this enzyme in the calcification process. We suggest

that this activity may be due a protein, which shares

common features with prokaryotic CA. This protein

shows similar features in the tissues and in the organic

matrix suggesting that the calcifying tissues could be

responsible for the secretion of this protein. This result

does not exclude the possibility that other types of CA

are responsible for the activity observed in the tissue

and the organic matrix. Our results also demonstrate

that in corals, organic matrix proteins are not only

structural proteins but also catalytic proteins and pro-

vide a crucial enzyme to eliminate the kinetic barrier in

the conversion of inorganic carbon. This new under-

standing of the chemistry in the calcifying region is

essential to account for the mechanisms underlying the

carbon and oxygen isotope fractionations seen in

skeletal carbonates (Adkins et al. 2003).

Acknowledgments We thank Prof. Francois Morel fromPrinceton University and Mak Saıto from the Woods HoleOceanographic Institution for providing the antibody, anti-b-carbonic anhydrase from Synecchococcus sp. This study wasconducted as part of the Centre Scientifique de Monaco 2000–2004 research program. It was supported by the Government ofthe Principality of Monaco and by the California Institute ofTechnology, USA.

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