A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains: A dual domain carbonic anhydrase

A novel carbonic anhydrase from the giant clam Tridacnagigas contains two carbonic anhydrase domainsWilliam Leggat1,2, Ross Dixon1, Said Saleh1 and David Yellowlees1

1 Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland, Australia

2 Centre for Marine Studies, University of Queensland, Queensland, Australia

Carbonic anhydrase (CA; EC 4.2.1.1) catalyses the

hydration of CO2 to HCO3–, is ubiquitous amongst all

living organisms and fulfils a variety of metabolic roles

[1]. Currently there are five evolutionarily distinct CA

gene families (a, b, c, d and e) [1,2] and it is generally

believed that all animal CAs belong to the a-CA fam-

ily. a-CAs are characterized by 36 amino acids found

around the active site [3]. Of these, 15 are conserved in

all active CAs, suggesting that they are required for

CA activity [3].

To date 15 a-CA or a-CA-like proteins have been

identified in mammals. These can be divided into five

broad subgroups, the cytosolic CAs (CA I, CA II,

CA III, CA VII and CA XIII), mitochondrial CAs

(CA VA and CA VB), secreted CAs (CA VI), mem-

brane-associated CAs (CA IV, CA IX, CA XII and

CA XIV) and those without CA activity, the

CA-related proteins (CA-RP VIII, X and XI). The

cytosolic and mitochondrial CAs and the secreted

and membrane-associated CAs are often further

Keywords

carbonic anhydrase; clam; symbiosis

Correspondence

W. Leggat, Centre for Marine Studies,

University of Queensland, Queensland

4072, Australia

Fax: +61 7 33654755

Tel: +61 7 33469576

E-mail: [email protected]

Notes

The nucleotide sequences for carbonic

anhydrase from T. gigas have been

deposited in the GenBank database under

GenBank accession numbers AY790884 and

AY799986-AY799998.

The alignment for the genomic sequence of

tgCA between positions 101 and 1810 of

the cDNA has been submitted to

EMBL-ALIGN database under the accession

number ALIGN_000833.

(Received 14 February 2005, revised

11 April 2005, accepted 28 April 2005)

doi:10.1111/j.1742-4658.2005.04742.x

This report describes the presence of a unique dual domain carbonic

anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important

role in the movement of inorganic carbon (Ci) from the surrounding sea-

water to the symbiotic algae that are found within the clam’s tissue. One of

these isoforms is a glycoprotein which is significantly larger (70 kDa) than

any previously reported from animals (generally between 28 and 52 kDa).

This a-family CA contains two complete carbonic anhydrase domains

within the one protein, accounting for its large size; dual domain CAs have

previously only been reported from two algal species. The protein contains

a leader sequence, an N-terminal CA domain and a C-terminal CA

domain. The two CA domains have relatively little identity at the amino

acid level (29%). The genomic sequence spans in excess of 17 kb and con-

tains at least 12 introns and 13 exons. A number of these introns are in

positions that are only found in the membrane attached ⁄ secreted CAs. This

fact, along with phylogenetic analysis, suggests that this protein represents

the second example of a membrane attached invertebrate CA and it con-

tains a dual domain structure unique amongst all animal CAs characterized

to date.

Abbreviations

CA, carbonic anhydrase; Ci, inorganic carbon; GPI, glycosylphosphatidylinositol.

FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3297

consolidated into two separate groups based upon

sequence similarity [3]. Of the membrane-associated

CAs, CA IX, XII and XIV contain integral trans-

membrane domains while CA IV is membrane atta-

ched through a glycosylphosphatidylinositol (GPI)

anchor.

Mammalian CAs function in a variety of roles inclu-

ding pH balance, H+ secretion, HCO3– secretion, CO2

exchange, bone resorption and ion transport [1]. The

diversity in mammalian genes and the presence of

homologs in other animals suggests that a large num-

ber of CAs are yet to be characterized from the animal

kingdom. Complete cDNA sequences from inverte-

brate sources have only been obtained from the tube-

worm Riftia pachyptila [4], cnidarians [5] and

mosquitoes [6,7]. In addition a cDNA sequence enco-

ding a protein involved in calcification from the pearl

oyster Pinctada fucata contains an a-CA-like domain

[8].

All animal CAs purified to date have subunit

molecular weights less than 58 kDa. The only excep-

tion is a report of the purification of a protein display-

ing CA activity from the giant clam Tridacna gigas,

where the purified protein has a molecular weight

of 70 kDa [9]. This protein is glycosylated through

both N- and O-links and is thought to be involved in

the transport of inorganic carbon (Ci) from seawater

to symbiotic photosynthetic dinoflagellates that are

found intercellularly within the clam tissue [9,10]. It

has been demonstrated that these alga can supply

up to 100% of the clam’s energy requirements [11].

In addition T. gigas contains two other CA isoforms,

one of 32 kDa and one of approximately 200 kDa

[9,10].

Here we present further characterization of the

70 kDa CA isoform from T. gigas, including cDNA

sequence indicating that it codes for a unique animal

CA containing two putative CA catalytic domains

within the one protein. Each domain contains those

residues thought to be essential for CA activity.

Results

tgCA cDNA sequence, deduced amino acid

sequence and domains

Only cDNA sequence for the first 1438 bp of the

70 kDa CA could be obtained from both the gill and

mantle cDNA libraries; both sequences were identical.

In both cases the sequence terminated in an identical

position (1438 bp after the start codon), suggesting that

the mRNA secondary structure prevented complete sec-

ond strand synthesis. For this reason cDNA sequence

was also obtained using RT-PCR using a higher than

normal temperature for second strand synthesis. Using

RT-PCR the 3¢ end of the cDNA was obtained yielding

an open reading frame of 1803 bp encoding 600 amino

acids and a protein of 66.7 kDa (Fig. 1). This is flanked

by a 58 bp 5¢-UTR and an 89 bp 3¢-UTR. Although no

polyadenylation signal was found a poly A tail was

sequenced. The translated protein sequence (tgCA) was

found to contain three domains, based upon database

searches, a signal sequence (Met1–Ala17) and two

domains with homology to the a-CA family, n-tgCA

(Ala18–Thr289) and c-tgCA (Ala290–Ser600) (Fig. 2).

The predicted cleavage point of the signal sequence,

between Ala17 and Ala18 (Centre for Biological

Sequence Analysis Database), produces a mature

N-terminal amino acid sequence almost identical (21

out of 22 residues) to an N-terminal peptide sequence

previously obtained [9], suggesting that this is the cor-

rect cleavage point for the signal sequence. A potential

GPI-modification site was identified at Gly577 by the

DPGI database.

Five consensus sites for N-glycosylation (NXS or

NXT) were found in the deduced amino acid sequence

at positions Asn66, Asn97, Asn177, Asn421 and Asn452.

Phylogenetic comparison of both CA domains with

a number of characterized human CA isoforms and

representative invertebrate CAs shows the clear group-

ing of the three recognized a-CA groups, the cytosolic,

Fig. 1. Translated protein sequence of tgCA

from T. gigas. The first CA domain (n-tgCA)

begins at Ala18, the second CA domain

(c-tgCA) begins at Ala290 (bold). The signal

sequence is highlighted and the five poten-

tial glycosylation sites (three in n-tgCA and 2

in c-tgCA) are underlined. Gly577 (double

underline) is the predicted GPI-anchor site.

A dual domain carbonic anhydrase W. Leggat et al.

3298 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS

secreted ⁄membrane-associated and the CA-related pro-

teins (Fig. 3). Both domains of the clam CA group

with the membrane-associated CAs.

Intron ⁄exon mapping

There are a number of intron ⁄ exon locations that are

specific for the various CA classes [3,12]. With this in

mind the introns for tgCA were mapped to further

characterize the two CA domains. The genomic

sequence of tgCA, between positions 101 and 1810 of

the cDNA, was amplified in a number of PCR reac-

tions spanning in excess of 17 kb. These sequences

included the complete coding sequence for the gene

between positions 101 and 1810. Twelve introns and

13 exons were found in this region, five in n-tgCA, six

Fig. 2. Alignment of n-tgCA and c-tgCA with other CAs showing intron position and conserved motifs. The 15 amino acids thought to be

required for CA activity are indicated (#), while cysteine residues involved in disulfide bonding in CA IV (two disulfide bonds) and CA VI, XII,

XIV (one disulfide bond) are indicated (�) above the alignment. Numbers below the alignment indicate the intron number while intron posi-

tions are represented by: ( ⁄ ) intron between amino acids, (\) intron located after the first codon position of the following amino acid, (+)

intron located after the second codon position of the following amino acid, and (*) represents no intron present. Residues shared by more

than 50% of the CAs examined are shaded. The alignment was performed using CLUSTALW. Note that hCA1 contains an alanine at position

122 rather then the conserved Val122, however, the consensus for the CA-1 isoform from vertebrates is valine [3]. hCA, human CA; NCBI

accession numbers in brackets, hCA1 (NM_001738), hCA2 (NM_000067), hCA3 (NM_005181), hCA4 (NM_000717), hCA5 (NM_001739),

hCA6 (NM_001215), hCA7 (NM_005182), hCA8 (NM_004056), hCA12 (NP_001209), hCA14 (NP_036245).

W. Leggat et al. A dual domain carbonic anhydrase

FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3299

in c-tgCA and one between the two domains (Fig. 2).

Unfortunately, despite repeated attempts it was not

possible to obtain genomic sequence corresponding to

the cDNA sequence prior to position 101 of the cDNA

sequence, this may have been due to the presence of

an extremely large intron. Alignment of the intron

positions with those of the human CAs shows that

both n-tgCA and c-tgCA share the majority of intron

locations with the secreted ⁄membrane-associated CAs

(Fig. 2). All introns conformed to the gt-ag rule [13]

(Table 1). In three cases (introns 2, 7 and 11) multiple

sequences were obtained for introns, suggesting that

tgCA is a multiple copy gene, this was confirmed by

the Southern analysis (data not shown). Only one base

pair of the genomic sequence differed to that previ-

ously obtained for the cDNA sequence (1586CfiT). Of

the � 15.7 kb of intron in the gene, sequence data was

obtained for � 9.8 kb. The GC content of the coding

sequence (44.5%) was significantly higher than that of

the introns (36.0%). In addition microsatellite repeat

sequences were found in exon 5 (CAAA, 21 repeats)

and exon 7 (GTTT, 13 repeats) (data not shown).

tgCA subunit size

In addition to the 70 kDa CA, another protein of

200 kDa with characteristics of CA was also identified

[9]. Although a minor component of the purified CA

fraction this protein had an identical N-terminal amino

acid sequence to the 70 kDa isoform [9]. When gel

purified and separated on an 8% SDS ⁄PAGE gel it

was found that the apparent molecular mass of this

Fig. 3. Phylogeny of the CA domains of

tgCA (n-tgCA and c-tgCA) with representa-

tives of other CA classes using maximum

likelihood. Alignments were performed using

CLUSTALW, bootstrapped 1000 times and the

trees constructed using maximum likelihood.

hCA, human CA; ce, Caenorhabditis

elegans; Dr, Drosophila melanogaster; ae,

Anthopleura elegantissma; CAH1 from the

alga Clamydomonas reinhardtii was used as

an outgroup.

Table 1. Exon ⁄ intron junctions of tgCA. No genomic sequence was obtained before the position corresponding to base pair 101 in the cDNA

sequence. Numbering of the cDNA sequence begins at the first codon position of the first in-frame methionine. The exact intron size is

given where known; estimates were made from the size of PCR products. Uppercase letters indicate coding sequence, while lowercase

indicate intron sequence.

Intron cDNA codon position preceding intron (bp) Intron size (bp) 5¢ Splice donor 3¢ Splice acceptor

1 265 � 1040 CACGG gtaaac ttccag TGGTA2 417 � 956a TTGAG gtaggt ttgtag GTACA3 510 � 1200 TTGAG gtgggt ttctag ATCGA4 583 � 1830 TAAAA gttagt ttctag ATGGA5 744 � 2280 CTCAG gtatat tttcag CTTGC6 868 � 2000 TACAG gttggg ttacag CTCAA7 910 � 1380a TCAAG gtatgt ttacag GAGTG8 1093 � 950 TACAA gtactt ctacag TCCAA9 1242 � 1100 TCGAG gtactg tttcag CTACA10 1335 � 1370 TTGAA gtaagt tttcag ATCGG11 1399 � 1300, �1200a TAAAG gtatgt tttcag ATGCC12 1581 347 GTCAG gtaagt ttccag TTGGT

a Two intron sequences were found for these introns.

A dual domain carbonic anhydrase W. Leggat et al.

3300 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS

protein is approximately 145 kDa, as opposed to

200 kDa suggested in [9]. In addition a protein of

70 kDa was also observed (Fig. 4A). When purified

CA containing the 32, 70 and 145 kDa isoform was

separated in the presence of increasing concentrations

of the reducing agent 2-mercaptoethanol, it was found

that the 145 kDa isoform disappeared (Fig. 4B). These

two observations, the presence of the 70 kDa isoform

in gel purified 145 kDa extract, and the disappearance

of the 145 kDa isoform under reducing conditions, in

addition to the identical N-terminal acid sequences [9]

suggests that the 70 kDa forms a homodimer of

� 145 kDa.

Separation of affinity purified CA by 2D-PAGE

shows that both the 32 and 70 kDa CAs have multiple

isoforms with identical masses but differing pI values

between 4 and 4.5 for the 32 kDa isoform and between

5.2 and 6.0 for the 70 kDa isoform (Fig. 5). The pre-

dicted pI point of the mature protein derived from the

cDNA sequence is 5.84.

Discussion

The data presented here represents the first example of

an animal protein containing two CA catalytic domains

within the one coding sequence. Only two other pro-

teins have been found to contain transcripts containing

duplicate CA domains which are translated into the

one peptide, one from the green alga Dunaliella salina

contains two a-CA domains while the second from the

red alga Porphyridium purpureum contains two b-CArepeats.

tgCA contains a 17 amino acid leader sequence, an

N-terminal CA domain of 272 amino acids and a

C-terminal domain of 311 amino acids. The cDNA

encodes a protein of 66.7 kDa, when the leader

sequence is removed the mature molecular mass is

reduced to 64.8 kDa, this is similar to the 62 kDa

molecular weight obtained for the deglycosylated pro-

tein determined by SDS ⁄PAGE [9]. Both CA domains

of tgCA contain all residues thought to be required for

CA activity [3] suggesting that both domains are cata-

lytically active. In addition, both domains contain a

histidine residue (His87 in n-tgCA, His363 in c-tgCA)

conserved with His64 in human CA2 (Fig. 2). This his-

tidine residue has been found to act as a proton shuttle

in CO2 hydration in high activity CAs (reviewed in

[14,15]) supporting the notion that both CA domains

within this protein are catalytically functional. How-

ever this will have to be confirmed experimentally

through either mutational studies or the use of select-

ive inhibitors.

The predicted cleavage position of the leader

sequence between Ala17 and Ala18 produces a mature

N-terminal protein sequence which is identical for the

first 21 amino acids to that obtained from N-terminal

sequencing of the purified protein. The putative signal

sequence for tgCA contains two potential in-frame

start codons (Met1 and Met3), similar to that found

in the membrane-associated human CA4 (Fig. 2). The

presence of this signal sequence is consistent with pre-

vious localization studies that have indicated extracel-

lular localization of this protein in both the mantle

and gills of giant clams [9,10].

Previous studies on the purified tgCA protein [9]

and immunolocalization results [9,10] suggest that

tgCA is a membrane CA, although its mode of attach-

ment was not clear. It was also found [9] that

A B

Fig. 4. SDS ⁄ PAGE separation of the various 70 kDa CA isoforms.

(A) An 8% SDS ⁄PAGE gel showing the presence of both the

145 kDa and 70 kDa CA isoforms in a purified extract of the

145 kDa isoform. Lane 1, purified 145 kDa CA isoform; lane 2, puri-

fied 70 kDa isoform. (B) Purified gill CA separated by SDS ⁄ PAGE in

the presence of increasing concentrations of 2-mercaptoethanol.

Lane 1, 0.25 M 2-mercaptoethanol; lane 2, 1.3 M 2-mercaptoetha-

nol; lane 3. 5.3 M 2-mercaptoethanol.

Fig. 5. Two-dimensional electrophoresis separation of affinity puri-

fied T. gigas CA. pI values are shown across the top while

molecular masses are shown on the right. Box 1 surrounds the

70 kDa isoform while box 2 surrounds the 32 kDa isoform.

W. Leggat et al. A dual domain carbonic anhydrase

FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3301

phosphoinositol phospholipase C digestion did not

result in the release of tgCA from crude gill homogen-

ate, suggesting that it is not GPI-anchored and is

instead an integral membrane protein. However analy-

sis of the predicted protein sequence presented here

does not seem to support this hypothesis, as evidence

of a putative transmembrane domain region is ambigu-

ous (data not shown). In addition the DPGI database

predicts that a GPI anchor may be present at Gly577

(it must be stated that the big-PI Predictor did not

identify a GPI-anchor for this protein). How tgCA is

associated with the membrane is still unknown and

requires further research, although the lack of a hydro-

phobic domain suggests that, as with human CA4, it is

attached through a GPI-anchor.

Perhaps surprisingly there is very little identity

between the two CA domains of this protein at either

the coding (48%) or amino acid level (29%). This level

of identity is similar to that seen when comparing the

different domains of tgCA to human CAs. Of the ver-

tebrate and invertebrate CAs used for the tree con-

struction (Fig. 3), n-tgCA had the greatest identity at

the amino acid level with human CA2 and human

CA7 (32% for both) while c-tgCA was most similar to

human CA1 and human CA7 (28% identity for both).

The lowest identity was with the alga C. reinhardtii

CAH1 gene (13% n-tgCA and 16% c-tgCA).

Despite this greater identity with human cytosolic

CAs, phylogenetic analysis suggests that both the

N- and C-terminal domains belong to the secreted ⁄membrane-associated CA group (Fig. 3). Despite low

bootstrap values for the tree in general, the support

for the division between the cytosolic and secre-

ted ⁄membrane-associated CAs is high (80%). This tree

clearly groups the human cytosolic CAs, the CA-like

proteins and secreted ⁄membrane-associated CAs. Of

the invertebrate CAs, both the fly and anemone fall

within the cytosolic group while the two putative

Caenorhabditis elegans CAs (CAH1 and CAH2) group

with the CA-RP vertebrate genes. To our knowledge

there is only one published report of a cDNA coding

for an invertebrate membrane attached CA [7], indica-

ting that tgCA represents the second example of a

membrane-associated CA from the invertebrates.

The phylogenetic grouping of tgCA with the mem-

brane-associated CAs from vertebrates is supported by

a range of other properties of this protein including

the presence of a signal sequence and the presence of a

conserved disulfide bond. All CAs of this group have

been found to contain two conserved cysteine residues

involved in an intrachain disulfide bond (Fig. 2). The

tgCA sequence is no exception with each CA domain

containing two conserved cysteine residues (Cys41,

Cys229, Cys315, Cys508) that are homologous to those

found in all other CAs of this group (Fig. 2). The pres-

ence of disulfide bonds in tgCA is supported by chan-

ges in electrophoretic mobility when the purified

protein is subjected to varying levels of reducing agent.

In the presence of higher concentrations of 2-merca-

ptoethanol, the 70 kDa isoform migrates more slowly

(Fig. 4A). This is consistent with human CA4 which

shows a similar pattern in the presence of 5% 2-merca-

ptoethanol [16].

While four of the six cysteines in tgCA are implica-

ted in intrachain disulfide bonds, evidence suggests

that at least one of the remaining two cysteines forms

a disulfide bond with another tgCA subunit making a

dimer of the 70 kDa protein. Gel filtration experiments

had previously suggested that tgCA exists as a dimer,

with a native weight of approximately 141 kDa [9].

When a gel purified fraction of similar estimated mole-

cular mass (145 kDa) is separated by SDS ⁄PAGE, the

70 kDa band is found in addition to the original

145 kDa protein. Upon addition of high concentra-

tions of the reducing agent 2-mercaptoethanol this

band (� 145 kDa) disappears (Fig. 4B) supporting the

conclusion that this represents a dimer. The gel filtra-

tion results in combination with reduction of the

145 kDa protein to the 70 kDa isoform suggest that

there are interchain disulfide bonds between two

70 kDa subunits.

Analysis of the genomic sequence data, where differ-

ent intron sequences have been obtained (Table 1),

Southern blots (data not shown) and protein two-

dimensional gels all suggest that tgCA is a multicopy

gene. Despite this no sequence differences were

observed in the coding sequence or intron position

where different copies, evidenced by different intron

sequences, were obtained. Given this it seems reason-

able then to use the combined genomic data, even if it

does not represent one gene, for analysis of intron ⁄exon structure.

Intron ⁄ exon positions are considered diagnostic for

animal CAs with characteristic pattern differences

being found in cytosolic and secreted ⁄membrane-asso-

ciated CAs [3,12]. For example of the 15 possible

intron sites shown in Fig. 2, three introns are shared

between these two groups, three are found only in the

cytosolic CAs and at least two more are found only in

the secreted ⁄membrane-associated CAs. Genomic

sequence of tgCA revealed 12 introns and 13 exons

(Fig. 2), all of which conformed to the gt ⁄ ag rule for

splice junctions [13] (Table 1). The majority of these

introns (11) were found to be homologous to those in

the secreted ⁄membrane-associated CAs (intron posi-

tions 5, 9 and 13 of all possible introns, Fig. 2) or

A dual domain carbonic anhydrase W. Leggat et al.

3302 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS

those introns common to the majority of vertebrate

CAs (introns 7, 8, 11). This distribution of intron ⁄ exonboundaries supports the phylogeny and protein prop-

erties discussed above that groups both CA domains

of tgCA with the secreted ⁄membrane attached CAs.

However, surprisingly one intron in the c-tgCA was

found to differ from this pattern. Intron 3 in c-tgCA

(Fig. 2) is diagnostic for the cytosolic CAs. The pres-

ence of a cytosolic specific intron in a CA that would

otherwise belong to the secreted ⁄membrane attached

CA grouping suggests that this intron was present

before the division of these two groups and has subse-

quently been lost from the membrane-associated CAs.

The dual domain structure of tgCA could have arisen

through one of two mechanisms, either the fusion of

two separate CA genes or a duplication of a single gene

followed by a fusion event. If this protein arose through

duplication and fusion event, and given the poor iden-

tity between the two CA domains (29%), the duplication

event must be old, thereby allowing time for the two

domains to diverge. This low identity between domains

is especially striking when compared to other duplicated

domain CA proteins, 52 and 72% identity for D. salina

and P. purpureum, respectively [17,18]. Furthermore

there are similar examples of non-CA duplicated domain

proteins from invertebrates. Phosphagen kinases from a

number of bivalves [19–21], sea anemones [22] and sea

urchins [23] have been shown to contain bi- or tripartite

repeat domains. In all of these examples, identity

between the domains is in excess of 60%. For each pro-

tein it has been concluded that the dual domain struc-

ture arose through gene duplication of one gene and

subsequent fusion. Where genomic sequence is available

[20,22] this is supported by the presence of an intron

between the two domains. Similarly in tgCA an intron is

found between the two domains. Given the low homol-

ogy between the two domains of tgCA in comparison to

other duplicated domain proteins it is not possible

to exclude the possibility that this protein has arisen

through the fusion of two different CA genes rather

than a duplication event.

Given the unique structure of this CA protein it

would be interesting to know if both domains display

CA activity. Previous studies [9] have shown that the

purified protein is active, however, from this data it is

not possible to conclude if this is due to one or both

domains. As both domains contain all the required

residues it seems likely that they are both active. A fur-

ther area of study is the interaction of the two

domains, for example are both required for activity

and ⁄or do they function cooperatively and what is

their three-dimensional arrangement? These questions

are areas of future study.

To date the dual domain structure of tgCA is unique

amongst animals, whether this gene duplication of CA

is present in other symbiotic or nonsymbiotic bivalves,

and possibly other invertebrates, remains to be seen. If

this CA arrangement is restricted to symbiotic bivalves

it may represent a mechanism by which a symbiotic

animal can increase the rate of inorganic carbon trans-

port to their photosynthetic symbionts, and thereby

maximize the benefits of symbiosis.

Experimental procedures

Purification of carbonic anhydrase from clam gills

The 70 kDa CA isoform was purified from the gills of

the giant clam T. gigas as previously described [9]. The

145 kDa CA isoform was electroeluted from affinity puri-

fied CA after separation by SDS ⁄PAGE.

Separation of CA isoforms by two-dimensional

gel electrophoresis

Affinity purified CA was analyzed by two-dimensional gel

electrophoresis (2D-PAGE). Separation in the first dimen-

sion was performed using an Immobiline DryStrip (pH 4–7,

Pharmacia, Piscataway, NJ, USA) which was then further

separated on an 8–18% SDS ⁄PAGE gradient gel using the

manufacturer’s protocol (Pharmacia, Cat # 18-1038-63).

Gels were visualized using Sypro-Ruby (Molecular Probes,

Eugene, OR, USA).

Purification of RNA and cDNA library construction

Total RNA was prepared from T. gigas mantle and gill

tissue. Fresh tissue (1.3 g) was snap frozen in liquid nitro-

gen and ground in a mortar and pestle. Total RNA was

prepared from the tissue using cesium chloride [24].

mRNA was then purified from total RNA using the

QuickPrep� mRNA Purification Kit (Pharmacia). The gill

cDNA was synthesized for rapid amplification of cDNA

ends by the polymerase chain reaction (RACE-PCR) using

the ClontechTM cDNA Amplification Kit. The mantle lib-

rary was initially synthesized as a phage library in k-ZapII(Stratagene, La Jolla, CA, USA). It was used as a

template for RACE-PCR using specific primers for the

adaptors.

Clam CA primers were designed to previously known

cDNA sequence of the 70 kDa CA isoform from T. gigas

[25] and to N-terminal amino acid sequence [9] (Fig. 1).

From the derived sequence further primers were designed

to amplify the remaining portion of the cDNA.

Products were also amplified using RT-PCR. mRNA was

purified as previously described and first strand synthesis

performed using OmniscriptTM Reverse Transcriptase

W. Leggat et al. A dual domain carbonic anhydrase

FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3303

(Qiagen, Valencia, CA, USA) using the following primer:

5¢-CCAgTgAgCAgAgTgACggAggACTCgAgCTCAAgCTT

TTTTTTTTTTTTTT-3¢. PCR products were then amplified

using a gene specific primer and Q0 (5¢-CCAgTgAgCAgAg

TgACg-3¢) whose sequence was contained in the poly(T)

primer. The second-strand synthesis was conducted at 38 �Crather than 16 �C to overcome problems associated with sec-

ondary structure inhibition of second-strand synthesis which

had previously been observed.

DNA sequencing

Gel purified PCR products (High Pure PCR Product Purifi-

cation Kit, Roche, Mannheim, Germany) or 1.5 lL of the

PCR reaction were ligated into T-Vector Easy (Promega,

USA) and transformed into XL-1 Blue cells. After plasmid

purification (High Purity Plasmid Isolation Kit, Roche)

clones were sequenced using capillary separation on a ABI

3730xl sequencer using the ABI v3.0 sequencing kit (Applied

Biosystems, Foster City, CA, USA).

Sequence analysis

Sequence alignments were performed using the program

clustal w [26], bootstrapped 1000 times and trees con-

structed using maximum likelihood [27], the C. reinhardtii

a-CA gene (CAH1) was used as an outgroup. All analyses

were performed using biomanager by ANGIS (http://

www.angis.org.au). Signal sequences were identified using

the Centre for Biological Sequence Analysis database [28].

Potential GPI-anchor sites were examined using the DPGI

database (http://129.194.185.165/dgpi/DGPI_demo_en.html)

and the big-PI Predictor (http://mendel.imp.univie.ac.at/gpi/

gpi_server.html) [29].

Isolation of genomic DNA

Genomic DNA was isolated from T. gigas sperm. Spawning

was induced by the injection of approximately 5 mL of a

2 mm serotonin solution into the gonads. The sperm was

collected from the water and centrifuged (1000 g for

5 min). Sperm (1 mL packed cell volume) was diluted with

11 mL proteinase K solution [50 mm Tris ⁄HCl pH 7.5,

20 mm EDTA, 100 mm NaCl, 1% (w ⁄ v) SDS, 100 lgÆmL)1

proteinase K] and the sample was incubated overnight at

55 �C. The solution was centrifuged (1000 g for 15 min at

4 �C), the supernatant removed, mixed with 10 mL of

ultra-pure phenol (Sigma, St Louis, MO, USA) and then

equilibrated with 4 mL of TE (10 mm Tris pH 8.0, 1 mm

EDTA) buffer. After adding an equal volume of chloro-

form, the solution was left overnight. The solution was

again centrifuged (1000 g for 15 min at 4 �C) and the aque-

ous phase removed. This was re-extracted twice with chlo-

roform and the DNA precipitated with 0.1 volume sodium

acetate (3 m, pH 5.2) and 2.5 volume 100% (v ⁄ v) ethanol.

After precipitation the DNA was spooled and resuspended

in TE buffer.

Genomic sequencing

The intron ⁄ exon structure of the 70 kDa CA was mapped

using a series of sequence specific primers obtained from the

cDNA sequence that bracketed possible intron positions [3].

Acknowledgements

This work was supported by an Australian Research

Council grant to David Yellowlees. We would like to

thank three anonymous referees for their helpful com-

ments.

References

1 Pastorekova S, Parkkila S, Pastorek J & Supuran CT

(2004) Carbonic anhydrases: current state of the art,

therapeutic applications and future prospects. J Enz

Inhib Med Chem 19, 199–229.

2 So AKC, Espie GS, Williams EB, Shively JM, Hein-

horst S & Cannon GC (2004) A novel evolutionary line-

age of carbonic anhydrase e is a component of the

carboxysome shell. J Bacteriol 186, 623–630.

3 Hewett-Emmett D & Tashian RE (1996) Functional

diversity, conservation, and convergence in the evolu-

tion of the a-, b-, and c-carbonic anhydrase gene famil-

ies. Mol Phylo Evol 5, 50–77.

4 De Cian M, Bailly X, Morales J, Strub J, van Doressel-

aer A & Lallier FH (2003) Characterization of carbonic

anhydrases from Riftia pachyptila, a symbiotic inverte-

brate from deep-sea hydrothermal vents. Proteins Struct

Func Genet 51, 327–339.

5 Weis VM & Reynolds WS (1999) Carbonic anhydrase

expression and synthesis in the sea anemone Anthopleura

elegantissima are enhanced by the presence of dinoflagel-

late symbionts. Physiol Biochem Zool 72, 307–316.

6 Pilar Corena M, Seron TJ, Lehman HK, Ochrietor JD,

Kohn A, Tu C & Linser PJ (2002) Carbonic anhydrase

in the midgut of larval Aedes aegypti: cloning, localiza-

tion and inhibition. J Exp Biol 205, 591–602.

7 Seron TJ, Hill J & Linser PJ (2004) A GPI-linked car-

bonic anhydrase expressed in the larval mosquito mid-

gut. J Exp Biol 207, 4559–4572.

8 Miyamoto H, Miyashita T, Okushima M, Nakano S,

Morita T & Matsushiro A (1996) A carbonic anhydrase

from the nacreous layer in oyster pearls. Proc Natl Acad

Sci USA 93, 9657–9660.

9 Baillie B & Yellowlees D (1998) Characterization and

function of carbonic anhydrase in the zooxanthellae-

giant clam symbiosis. Proc R Soc Lond B 265, 465–473.

A dual domain carbonic anhydrase W. Leggat et al.

3304 FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS

10 Leggat W, Marendy EM, Baillie B, Whitney SM,

Ludwig M, Badger MR & Yellowlees D (2002) Dinofla-

gellate symbioses: strategies and adaptations for the

acquisition and fixation of inorganic carbon. Funct

Plant Biol 29, 309–322.

11 Fisher CR, Fitt WK & Trench RK (1985) Photosynth-

esis and respiration in Tridacna gigas as a function of

irradiance and size. Biol Bull 169, 230–245.

12 Jiang W & Gupta D (1999) Structure of the carbonic

anhydrase VI (CA6) gene: evidence for two distinct

groups within the a-CA gene family. Biochem J 344,

385–390.

13 Mount SM (1982) A catalog of splice junction

sequences. Nucleic Acids Res 10, 459–472.

14 Lindskog S (1997) Structure and mechanism of carbonic

anhydrase. Pharmacol Ther 74, 1–20.

15 Supuran CT, Scozzafava A & Casini A (2003) Carbonic

anhydrase inhibitors. Med Res Rev 23, 146–189.

16 Zhu XL & Sly WS (1990) Carbonic anhydrase IV from

human lung: purification, characterization, and compar-

ison with membrane carbonic anhydrase from human

kidney. J Biol Chem 265, 8795–8801.

17 Fisher M, Gokhman I, Pick U & Zamir A (1996) A

salt-resistant plasma membrane carbonic anhydrase is

induced by salt in Dunaliella salina. J Biol Chem 271,

17718–17723.

18 Mitsuhashi S & Miyachi S (1996) Amino acid sequence

homology between N- and C-terminal halves of a carbo-

nic anhydrase in Porphyridium purpureum, as deduced

from a cloned cDNA. J Biol Chem 271, 28703–28709.

19 Compaan DM & Ellington WR (2003) Functional con-

sequences of a gene duplication and fusion event in an

arginine kinase. J Exp Biol 206, 1545–1556.

20 Suzuki T, Kawasaki Y, Unemi Y, Nishimura Y, Soga

T, Kamidochi M, Yazawa Y & Furukohri T (1998)

Gene duplication and fusion have occurred frequently

in the evolution of phosphagen kinases – a two-domain

arginine kinase from the clam Pseudocardium sachalinen-

sis. Biochim Biophys Acta 1388, 253–259.

21 Suzuki T, Sugimura N, Taniguchi T, Unemi Y, Murata

T, Hayashida M, Yokouchi K, Uda K & Furukohri T

(2002) Two-domain arginine kinases from the clams

Solen strictus and Corbicula japonica: exceptional amino

acid replacement of the functionally important D62 by

G. Int J Biochem Cell Biol 34, 1221–1229.

22 Suzuki T, Kawasaki Y & Furukohri T (1997) Evolution

of phosphagen kinase: Isolation, characterization and

cDNA-derived amino acid sequence of two-domain

arginine kinase from the sea anemone Anthopleura japo-

nicus. Biochem J 328, 301–306.

23 Wothe DD, Charbonneau H & Shapiro BM (1990) The

phosphocreatine shuttle of sea urchin sperm: flagellar

creatine kinase resulted from a gene triplication. Proc

Natl Acad Sci USA 87, 5203–5207.

24 Sambrook J, Fritsch EF & Manniatis T (1989) Mole-

cular Cloning, 2nd edn. Cold Spring Harbor Laboratory

Press, Cold Spring Harbour, NY.

25 Baillie BK, (1995) Inorganic carbon acquisition and utili-

sation in the giant clam symbiosis. PhD Thesis, James

Cook University of North Queensland, Australia.

26 Thompson JD, Higgins DG & Gibson TJ (1994) CLUS-

TAL W: improving the sensitivity of progressive multi-

ple sequence alignment through sequence weighting,

position-specific gap penalties and weight matrix choice.

Nucleic Acids Res 22, 4673–4680.

27 Felsenstein J (1989) PHYLIP – Phylogeny Inference Pack-

age Version 3.2. Cladistics, 5, 164–166.

28 Bendtsten JD, Nielsen H, von Heijne G & Brunak S

(2004) Improved prediction of signal peptides: SignalP

3.0. J Mol Biol 340, 783–795.

29 Eisenhaber B, Bork P & Eisenhaber F (1999) Prediction

of potential GPI-modification sites in proprotein

sequences. J Mol Biol 292, 741–758.

W. Leggat et al. A dual domain carbonic anhydrase

FEBS Journal 272 (2005) 3297–3305 ª 2005 FEBS 3305