Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii

Fish & Shellfish Immunology (2000) 10, 343–357 doi:10.1006/fsim.1999.0244 Available online at http://www.idealibrary.com on

Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii MARIA ROSARIA COSCIA1*, VERONICA MOREA2,3, ANNA TRAMONTANO2 UMBERTO ORESTE1

AND

1

Institute of Protein Biochemistry and Enzymology, CNR, Via Marconi 10, 80125 Naples, Italy, 2IRBM ‘‘P. Angeletti’’, via Pontina Km 30, 00045 Pomezia, Rome, Italy, and 3Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, U.K. (Received 21 June 1999, accepted after revision 4 October 1999) A spleen cDNA library was constructed from the Antarctic teleost Trematomus bernacchii and immunoscreened with rabbit IgG specific for T. bernacchii Ig heavy chain. Eleven cDNA clones, varying in size and encoding the entire heavy chain or parts of it, were isolated. Here the complete nucleotide and deduced amino acid sequences of clone 2C2 encoding the secretory IgH chain form are reported. Comparison of the amino acid sequence of the entire constant region of the T. bernacchii Ig heavy chain with those from other teleosts and two holostean fish showed percent identity ranging 53·6–60·6%, with the highest values found for Salmoniformes. The multiple sequence alignment revealed the presence of two remarkable insertions: one at the VH-CH1 boundary and a second one, not found in any other IgM heavy chain, localised at the CH2-CH3 boundary. The latter occurred in the region proposed to act as a ‘hinge’, and resulted in a CH2–CH3 hinge peptide longer than any other IgM hinge. Di#erences were also found in the number and position of putative N-glycosylation sites of the compared sequences. It is suggested that the unusual features found in the T. bernacchii Ig heavy chain might contribute to the flexibility of the Ig molecule and help understand more about the adaptation of Ig molecules to the polar sea environment.  2000 Academic Press

Key words:

fish IgH chain, Nototheniidae, Ig hinge region, cold-adapted protein, polar fish, T. bernacchii cDNA library.

I. Introduction The most represented fish Ig has been identified as IgM on the basis of several physicochemical features, such as size of the H chain, carbohydrate content and polymeric assembly of the basic unit H2L2 (tetrameric in teleosts) (Wilson & Warr, 1992). Heavy chain genes from the most studied species belonging to the Teleostean orders Elopomorpha, Siluriformes, Gadiformes, Cypriniformes and Salmoniformes, e.g. Elops saurus (Amemiya & Litman, 1990), Ictalurus * Corresponding author. E-mail: [email protected] 1050–4648/00/040343+15 $35.00/0

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punctatus (Gha#ari & Lobb, 1989a, 1989b), Gadus morhua (Bengtén et al., 1991), Cyprinus carpio (Nakao et al., 1998) and Oncorhynchus mykiss (Lee et al., 1993) have been cloned and sequenced. These analyses revealed some structural characteristics of the teleost H chain. It is approximately 20 amino acid shorter than that of tetrapods. The position of invariant cysteine and tryptophan residues (Chothia et al., 1988) is conserved, but the number and distribution of extra cysteines, likely involved in inter-H chain and intersubunit linkages, as well as of putative N-glycosylation sites vary within the teleost group. Based on phylogenetic comparisons of the amino acid sequence, the teleost CH4 domain shows the highest similarity with the CH4 domain of higher vertebrates, whereas the CH3 domain shows the greatest diversity. Recently, the presence of two closely related CH genes (CHA and CHB) has been demonstrated in Atlantic salmon, Salmo salar (Hordvik et al., 1992, 1997). The study of fish species living in the Antarctic seas at about
2 C is of interest to shed light on evolutionary adaptation to cold conditions in terms of morphology, physiology and biochemistry. Most Antarctic ichthyofauna (34·7%) are of the perciform suborder Notothenioidei, which includes six families and 120 species (Gon & Heestra, 1990). Antarctic ichthyologists hypothesise that Notothenioidei arose 12–14 million years ago, adapting to the progressively colder conditions of the Southern Ocean; in particular the subfamily of Trematominae have been speciated 2·5–4·8 million years ago (Eastman, 1993). Over the last 30 years, the biochemical and physiological processes of several Notothenioidei species have been studied intensively in order to better understand how these processes enable life under the unique conditions of the Antarctic marine environment. Recently Igs have been purified from serum of T. bernacchii and other Antarctic fish species of the notothenioid families Channichthyidae and Nototheniidae (Coscia & Oreste, 1997), and their relative concentration has been determined (Scapigliati et al., 1997). The presence in plasma and secretions of Antarctic fish of naturally occurring antibodies specific for antigens of the nematode parasites Contracaecum osculatum and Pseudoterranova decipiens has been previously investigated (Coscia & Oreste, 1998a, 1998b). Analysis of putative structural features accounting for the cold adaptation of IgM is of great interest. Polymeric IgM are the most complex Ig molecules, thought to be very flexible, particularly at the CH2–CH3 junction (Roux et al., 1998); attention has been focused on the C-terminal secretory tailpiece which has been highly conserved throughout evolution, carrying important glycans (de Lalla et al., 1998) and participating in the polymeric assembly (Davis et al., 1998). To approach the structure–function relationship of cold-adapted Ig we investigated the amino acid sequence of the H chain of Trematomus bernacchii IgM and compared it with other fish IgH. II. Materials and Methods COLLECTION OF TISSUES

Trematomus bernacchii specimens (300 g) were caught in the Ross Sea, near the Italian Station at Terra Nova Bay (7442 S, 16467 E). Blood samples were

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collected from several specimens by caudal venipuncture and pooled. After several hours at 4 C, sera were separated from the clot by centrifuging at 2000g for 10 min and stored at
20 C. Spleen was removed, immediately frozen in liquid nitrogen and stored at
80 C until used. PREPARATION OF RABBIT ANTI-IGH CHAIN ANTIBODIES

T. bernacchii Ig were purified from sera as previously described (Coscia & Oreste, 1997). The purified Ig were subjected to electrophoresis in a 12% SDS polyacrylamide slab gel run under reducing conditions. The H-chain band was then electroeluted from the gel for 5 h at 60 mA, dialysed against 50 mM phosphate bu#er, pH 7·2 containing 150 mM NaCl and used as immunogen to raise antisera against T. bernacchii IgH chain. Two New Zealand White rabbits (Charles River Kingston, NY, U.S.A.), named RbC and RbD, were each injected intradermally with 0·5 ml of 0·2 mg ml
1 immunogen emulsified with equal volume of Freund’s complete adjuvant. The animals were boosted intramuscularly five times biweekly with the same dose emulsified with Freund’s incomplete adjuvant. A week after the last injection, the rabbits were bled by cutting the marginal ear vein. After several hours at 4 C, sera were separated from the clot by centrifuging at 2000g for 10 min and stored at
20 C. Of the two rabbit antisera raised, the RbC antiserum showed the highest titre of T. bernacchii H chain-specific IgG by dot-blot immunoassay, performed as previously described (Coscia & Oreste, 1997). Antibodies were purified from the serum by using the MAbTrap G II kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) and then adsorbed to H chain coupled with CNBr activated-Sepharose 4B (Pharmacia Biotech) following the procedure described previously (Coscia & Oreste, 1998a). The specificity of the resulting purified rabbit anti-T. bernacchii IgH antibodies was tested following the ECL Western blotting protocol (Amersham International, Bucks, U.K.) by using a 1:1000 dilution of rabbit anti-T. bernacchii H chain IgG (8 ng ml
1) as the primary antibody and horseradish peroxidase labelled anti-rabbit antibodies as the secondary antibody, at the dilution recommended by the manufacturer. No cross-reaction of anti-H chain antibodies with the L chains blotted onto the same nitrocellulose membrane was observed. CONSTRUCTION OF AN IGH CHAIN cDNA LIBRARY

A T. bernacchii spleen (0·5 g) was thoroughly homogenised in a chilled homogeniser, and total RNA was isolated by the QuickPrep Total RNA extraction kit (Pharmacia Biotech) following the manufacturer instructions. The poly(A) + RNA was purified by conventional a$nity chromatography on an oligo (dT)-cellulose column (mRNA purification kit, Pharmacia Biotech) and 5
g was used for cDNA synthesis. The T. bernacchii cDNA library was constructed in ZAP Express vector using the ZAP Express cDNA Synthesis kit (Stratagene Cloning System, La Jolla, CA, U.S.A.) and packaged using the Gigapack III Gold Packaging Extract (Stratagene). The library contained 7105 recombinant plaques and, after amplification, 3108. The E. coli strain XL1-Blue was used as host for immunological screening of the expression

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library, following the isopropylthio--D-galactoside method as described by Sambrook et al. (1989). The primary screening of the library was carried out at a plaque density of 3104 plaques/150 mm plate and the following two at 1104 plaques/90 mm plate. Nitrocellulose filters (Schleicher and Schuell, Germany) were first incubated with rabbit anti-T. bernacchii IgH antibodies, preadsorbed with E. coli extract (0·1 mg ml
1) for 30 min at room temperature, and then in sequence with biotin-labelled goat anti-rabbit IgG (Kirkegaard and Perry Laboratories, MD, U.S.A.) and peroxidase labelled streptavidin (SP; Kirkegaard and Perry Laboratories). Antigen–antibody–antibody–SP complexes were detected using the chromogenic substrate 4-chloro-1-naphthol (HRP Color Development Reagent; BioRad Laboratories, Richmond, CA, U.S.A.). The developing solution was prepared mixing 2 ml of this substrate (3 mg ml
1 in methanol) with 123
l of 1 M imidazole (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 5
l of 30% H2O2 in 10 ml of 500 mM Tris, pH 7·9, containing 150 mM NaCl. After three consecutive immunoscreenings, several positive clones were identified and inserts cloned into the ZAP Express vector were excised out of the phage in the form of the kanamycin-resistant pBKCMV phagemid vector using the ExAssist helper phage and XLOLR strain in accordance with the manufacturer instructions (Stratagene). The size of the cDNA fragments was estimated by reference to DNA Molecular Weight Marker III (Boehringer Mannheim, Mannheim, Germany), performing agarose gel electrophoresis. DNA SEQUENCING

Immunopositive clones were sequenced in both directions according to the dideoxynucleotide chain termination method (Sanger et al., 1977) with T7 DNA polymerase (Pharmacia Biotech), using the plasmid sequencing primers T3 and M13–20 as well as synthetic oligonucleotides. SEQUENCE ANALYSIS

The nucleotide sequences were compared to the GenBank/EMBL databases by searching with the BLAST program (Altschul et al., 1990) and translated into amino acid sequences with the MacMolly program (Softgene GmbH, Germany). The deduced amino acid sequence was then compared to a nonredundant database of protein sequences (nr NCBI, 17 June 1998 database) and the database of sequences of known protein structures (PDB NCBI, 19 July 1998 database) by searching with the BLAST program (Altschul et al., 1990). The nucleotide and deduced amino acid sequences were analysed using the Genetics Computer Group Sequence Analysis Software Package Version 8·1-UNIX (Madison, WI, U.S.A.) (Devereux et al., 1984). Multiple alignments with complete amino acid sequences from the most studied teleost species and with the sequences of higher vertebrates were performed with the CLUSTAL W program (Thompson et al., 1994), version 1·7, using default parameters. The percent amino acid identity of T. bernacchii CH domains with those from other fish was calculated with the GAP program of the GCG package (Devereux et al., 1984). The amino acid composition was determined using the DNA Strider program (Marck, 1988).

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MODEL BUILDING AND ANALYSIS

Atomic models have been built for the VH sequence of T. bernacchii and for a sequence from O. mykiss (GenBank/EMBL accession number UO4616), using the canonical structures method for the hypervariable loops of the antigen binding site (Chothia & Lesk, 1987; Chothia et al., 1989; Al-Lazikani et al., 1997) and standard homology modelling techniques for the framework regions. In both cases the VH structure of the human IgG1 from myeloma (PDB entry: 8fab) (Bernstein et al., 1977) was used as template for the main-chain conformation of the framework regions. Sequence identity between the VH of 8fab and the VH domains of T. bernacchii and O. mykiss was 55 and 53%, respectively. The sequence identity between the VH domains of T. bernacchii and O. mykiss was 64%. In both models, the H1 loop was modelled using that of 8fab, which has the same canonical structure, as template for the mainchain conformation. The H2 and H3 loops of both sequences were imported from mouse immunoglobulin D1·3 (PDB entry: 1vfb) (Bernstein et al., 1977). III. Results IDENTIFICATION OF IGH cDNA CLONES

A cDNA library was constructed from the spleen of a T. bernacchii specimen using the ZAP Express vector and screened with RbC antibodies raised against T. bernacchii IgH-chain band electroeluted from an SDS-PAGE slab gel of purified fish Ig, run under reducing conditions. After three consecutive screenings of the library, 11 immunopositive clones were identified and sequenced. Seven clones varied in size and encoded di#erent parts of the C region. The other four clones were found to be of similar size (1·9 kb) and, based on overall length and on the presence of the poly-A tail, were considered to represent the full length transcript. These clones were expected to contain an internal Eco RI site since, when restricted, their inserts gave two fragments (1·3 and 0·6 kb). After determining the nucleotide sequence, the Eco RI site was identified at the end of the CH1 domain, coding for Glu 218 and Phe 219. ANALYSIS OF DEDUCED AMINO ACID SEQUENCE

The complete nucleotide and deduced amino acid sequences of the cDNA clone 2C2 are shown in Fig. 1. This clone (1937 bp) was found to encode the entire V region and the four C domains, including a 20 amino acid leader peptide, the polyadenylation signal, the poly-A tail and a 137-bp 3 untranslated region. The VH domain was shown to contain all of the canonical residues conserved in other teleosts and in most vertebrates (Chothia et al., 1988). Comparison of the VH sequence of T. bernacchii with the VH sequence of other teleosts, and with the consensus VH sequences of mammals, did not reveal any significant di#erence in their amino acid composition and hydrophobicity profiles. The three-dimensional models from T. bernacchii and O. mykiss (UO4616) were built to identify buried and exposed residues, and no di#erence in their composition with respect to hydrophilic or hydrophobic residues was observed (data not shown).

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Fig. 1. Complete nucleotide sequence of the T. bernacchii IgH chain cDNA clone 2C2. GenBank accession number AF094531. The deduced amino acid sequence is reported in one-letter code. The sequence was divided into leader peptide, CDRs, FRs and CH domains on the basis of sequence comparisons with the H chain of other teleosts. The internal Eco RI site is shown in bold and shaded. The polyadenylation signal is underlined. The putative N-glycosylation sites are designated in bold and underlined. A sequential numbering is used.

When the amino acid sequence of each T. bernacchii CH domain was compared with the respective teleost sequences (Table 1), the CH1 domain showed the highest identity (72·0%) with G. morhua, whereas the CH4 domain showed the highest identity (75·4%) with O. mykiss. The whole CH1–CH4

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Table 1. Percent amino acid identity between constant domains

I. punctatus G. morhua O. mykiss S. salar E. saurus A. calva L. osseus

CH1–CH4

CH1

CH2

CH3

CH4

56·1 57·2 60·6 58·6 55·3 54·5 53·6

59·0 72·0 67·7 67·6 60·2 63·2 59·4

43·6 44·5 42·3 45·8 46·7 48·1 49·5

51·5 46·2 54·2 52·0 54·1 47·3 47·2

62·7 61·9 75·4 73·8 59·2 58·4 58·4

Percent amino acid identity between the CH domains of T. bernacchii and the teleosts Ictalurus punctatus (Gha#ari & Lobb, 1989b), Gadus morhua (Bengtén et al. 1991), Oncorhynchus mykiss (Lee et al., 1993), Salmo salar (Hordvik et al., 1992), Elops saurus (Amemiya & Litman, 1990) and the two holostean species Amia calva and Lepisosteus osseus (Wilson et al., 1995). Percent identities were calculated using the GAP program of the GCG package.

region was also compared, and identity was found to be higher with Salmoniformes, ranging 57·2–60·6%, than with Siluriformes. In addition, the C region of the T. bernacchii IgH chain was compared with the sequences of two holostean fish, the bowfin, Amia calva, and the longnose gar, Lepisosteus osseus (Wilson et al., 1995), resulting in percent identities only slightly di#erent from those of the teleosts analysed. As can be seen in the alignment shown in Fig. 2, 64 residues were found to be conserved in the C region: the cysteines forming the intradomain disulfide bridges as well as those involved in the L-chain binding (Chothia et al., 1985), and the tryptophan residues, except that one at the end of CH2, which is not the tryptophan residue packing against the disulfide bond in the folded structure of the Ig domain (Chothia et al., 1985). Conversely, five substitutions, Phe/Ser, Arg/Gln in CH1, Phe/Leu, Trp/Pro in CH2 and Leu/Val in CH3, indicated by an open circle in Fig. 2 were observed. In particular, the tryptophan residue in CH2 represents an exception among the conserved residues; however, the poor alignment of this region did not allow determination of whether this residue is replaced by another or whether there is partial refolding in the region. A similar substitution (Trp/Phe) is observed in the CH1 domain of E. saurus. In addition, several insertions were noted that are unique to T. bernacchii with respect to the other teleost species (Fig. 2): one at the beginning of the CH1 domain, one in CH2 and two in CH3. Thus, in T. bernacchii, the area neighbouring the VH-CH1 switch region, the latter identified as residues 113, 114 and 115 (Padlan, 1994), contains two extra residues with respect to most of the other teleosts and between one and three extra residues with respect to the IgM sequences of higher vertebrates (data not shown). In IgH chains, the region where this insertion occurs is structurally close to the ‘ball and socket’ joint between the VH and CH1 domains that is involved in the ‘elbow motion’ of Igs, which is the movement of the VL-VH dimer relative to the CL-CH1 dimer (Lesk & Chothia, 1988) (Fig. 3). The ball and socket joint is formed by five residues almost absolutely

Fig. 2. Multiple alignment of the amino acid sequences of the VH and CH regions from I. punctatus, clone NG21 (GenBank accession number M58669), G. morhua, clone cSg 5·1 (GenBank accession number X58870), O. mykiss, clone cRTVH 50 (GenBank accession number S63348), S. salar, clone 7·3 (GenBank accession number Y12456), E. saurus, clone 14501 (GenBank accession number M26182) and T. bernacchii, clone 2C2 (GenBank accession number AF094531). Gap positions were manually optimised and are indicated by hyphens. Amino acid residues found identical in all of the compared sequences are in bold. Positions at which amino acid residues are di#erent in T. bernacchii are indicated by an open circle. The putative N-glycosylation sites and extra-cysteines are underlined. Positions usually involved in the ball and socket joint are indicated by the symbol +. Demarcations of the ‘‘CH2–CH3 hinge peptide’’ are located according to Padlan (1994). Position numbering is based on the convention of Kabat (1991).

350 M. R. COSCIA ET AL.

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Fig. 3. A typical ball and socket joint between the VH and CH1 domains of Igs (Lesk & Chothia, 1988). The backbone atoms of residues in the ball and socket region of the anti-TCR Fab fragment (PDB entry: 1nfd) (Bernstein et al., 1977) are shown (magenta) together with the side-chains of the residues forming the ‘ball’ and the ‘socket’ (green: carbon atoms, red: oxygen atoms, blue: nitrogen atoms). The region where an insertion is expected to occur in T. bernacchii is orange. The residues of the anti-TCR Fab fragment are numbered according to Kabat (1991). Residues present in T. bernacchii in the corresponding positions are in parentheses.

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conserved in the sequences of Igs and T-cell receptors (Chothia et al., 1985). In the T. bernacchii sequence four of these five residues (Thr 11, Thr 110, Thr 112 and Pro 149), indicated by the symbol + in Fig. 2 are compatible with the ball and socket mechanism, but the presence of Ala in position 148, where a larger hydrophobic residue (Phe or Leu) is usually observed in higher vertebrates (Lesk & Chothia, 1988) suggests that the elbow motion of this antibody might involve a di#erent mechanism and/or di#erent residues from those previously described. The sequence similarity is not su$ciently high to precisely locate the position of the other insertions, but their extent can easily be deduced by the spacing between conserved cysteines. The number of amino acid residues between the two conserved cysteines in CH3 was markedly di#erent in T. bernacchii (56 aa) than in I. punctatus (50 aa), G. morhua (49 aa), O. mykiss (50 aa), S. salar (49 aa) and E. saurus (48 aa). In addition, the region between the last conserved cysteine of CH2 and the first of CH3 is longer than in other teleosts (Fig. 2) and in the IgM sequences of higher vertebrates (data not shown). This portion of the T. bernacchii molecule can be viewed as a ‘hinge’ by comparison with the mammalian IgG, IgA and IgM hinges (Padlan, 1994). It contains two extra cysteines, which might be expected to participate in interchain disulfide linkages, and two prolines, providing rigidity to the central part of the ‘CH2–CH3 hinge’. This region is glycine rich and therefore could act as a flexible spacer. Most of the other non-glycine and non-proline residues are either hydrophilic or charged, suggesting that this region is solvent exposed. Other remarkable di#erences were observed in the putative N-glycosylation sites of the compared sequences. Two additional putative N-glycosylation sites, Asn-X-Ser/Thr (Marshall, 1972) were found in the CH2 domain, close to the ‘CH2–CH3 hinge’, while the site in CH3, in fish generally found at the end of the domain, was at a di#erent position. Four cysteine residues, not involved in intradomain bonds, were identified: one more than S. salar, O. mykiss, I. punctatus or G. morhua, but one less than E. saurus. Two of them, probably responsible for the H–L and H–H chain pairings, were found at conserved positions, in CH2 and C-terminus respectively; the remaining two were located in the ‘CH2–CH3 hinge’. The relative molecular mass calculated from the amino acid composition (Table 2) is 64·6 kDa. Comparing this value with that of the purified H chain determined by SDS-PAGE (75 kDa) (Coscia & Oreste, 1997), it might be concluded that the carbohydrate content is approximately 14%. IV. Discussion Sequence analysis and comparison with IgM heavy chain sequences from other teleost species enabled us to provide a picture of the structural features of Antarctic fish Igs. The T. bernacchii sequence presents novel features partly in common with higher vertebrates and not seen in other teleosts. The regions that allow the flexibility of mammalian antibody molecules are the switch region at the VH–CH1 boundary and involved in determining the elbow angle variability (Lesk & Chothia, 1988), and the hinge region (Padlan, 1994). The

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Table 2. Amino acid composition of the T. bernacchii CH region Amino acid ala cys asp glu phe gly(a) his ile lys leu met asn pro gln arg ser(b) thr(a) val(b) trp ty total

n

n (%)

21 12 24 30 16 33 8 24 33 33 8 25 27 17 14 31 53 31 8 16 464

4·5 2·6 5·2 6·5 3·4 7·1 1·7 5·2 7·1 7·1 1·7 5·4 5·8 3·7 3·0 6·7 11·4 6·7 1·7 3·4 100

Amino acid composition of the T. bernacchii CH region. (a) Residues found in higher or (b) lower number upon comparison with the amino acid composition of the CH region from G. morhua or O. mykiss or H. sapiens.

hinge region has been investigated in all of the mammalian isotypes. However, very little three-dimensional data are available, and thus there is incomplete knowledge of the structure. In IgG and IgA the hinge regions have been clearly located between the CH1 and the CH2 domains, giving the Fabs the freedom to rotate and the Fc the freedom to wag. Moreover, the hinge region and the interdomain areas between the VH and the CH1 domains of the Fabs have been shown to be the portions of IgG molecules where the main temperature-dependent conformational changes occur (Calmettes et al., 1991). Indeed, in IgM and IgE the hinge, supposed to be between the CH2–CH3 domains, has been studied less extensively. In the bowfin and gar an unusually long proline-rich region has been described and is proposed to act as a hinge region (Wilson et al., 1995). It is agreed that the length of the hinge ensures both the flexibility of the Fabs to cope with di#erent epitope orientations, and the accessibility of Fc to other molecules for its e#ector functions. Interestingly, the two regions that most influence the segmental flexibility of the molecule are longer in T. bernacchii H chain. The presence of two extra residues, presumably alanine and threonine, at the VH–CH1 boundary, might alter the structure locally and thereby might facilitate the elbow motion. The observation of two hexamer repeats found at the beginning of the CH1 domain

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leads to the suggestion that the insertion of the two residues alanine and threonine may possibly be the result of a nucleotide duplication. The particularly long hinge might allow the molecule to fix complement more e$ciently because the Fab arms are kept far enough apart not to cover the binding motif for C1q, HXDXXXP (Perkins et al., 1991), identified also in T. bernacchii at the end of CH3. Analysis of genomic DNA is needed to confirm that the additional nucleotides coding for a longer CH3 are in 5 of CH3 rather than downstream of CH2. Due to the negative hydropathic index of 13 out of 17 amino acid residues, the ‘CH2–CH3 hinge’ is expected to be on the exterior in the native protein, and thus to be the most susceptible portion of the molecule to proteolytic enzymes. The H chain was found to be heavily glycosylated; in particular the presence of at least four putative N-glycosylation sites within approximately 80 amino acid residues surrounding the ‘CH2–CH3 hinge’ suggests the carbohydrates protect from cleavage by proteases. The glycosylation site thought to be important for preventing dimerisation of the CH3 domain in mammalian IgM and IgE was not found at the end of the domain, as it is in I. punctatus, O. mykiss, S. salar and E. saurus, but rather at a di#erent position, similar to G. morhua, and the very conserved glycosylation site at the C-terminus was missing in T. bernacchii, as in G. morhua and E. saurus. This feature of the T. bernacchii C-terminus is certainly significant in the assembly of polymeric Ig. Comparison of the four separate CH domains with those from other teleosts (Table 1) confirms that the CH2 and the CH3 domains are more diverse than CH1 and CH4. The two holostean fish, the closest ancestors of the modern bony fish (teleosts), were also compared. However, no striking di#erence in the percent amino acid identity was found. Fifty-six amino acid residues were found between the two invariant cysteines in CH3. This number is unusually high compared with other teleosts but, interestingly, is closer to the number in mammals, e.g. there are 59 amino acids in the mouse. In T. bernacchii there are two cysteine residues at the beginning of the CH3 domain, where there are no cysteines in other teleosts, except for one in cod and ladyfish, located at the end of CH2. This finding is noteworthy since it suggests that these two cysteine residues in the middle of the T. bernacchii H chain may be involved in a double inter-H chain linkage, resembling the disulfide bonds described in variable number in human IgG hinges. In contrast, in channel catfish and salmon CHB, an unusual cysteine has been found in close proximity to the conserved C-terminal cysteine, hypothetically involved in the formation of several subpopulations of covalently linked structures according to various, changeable combinations of disulfide bonds (Gha#ari & Lobb, 1989a). Sequence analysis of a limited number of cDNA clones encoding the C domains did not reveal any amino acid di#erence. However, it would be of interest to verify the existence of allotypic variants, as described in other teleosts (Hordvik et al., 1992; Hansen et al., 1994), and of Ig isotypes other than IgM, e.g. IgD found in channel catfish (Wilson et al., 1997). The amino acid composition of the T. bernacchii CH region was found to be lower for serine and valine and higher for threonine and glycine in all of the comparisons both with other teleosts and with

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humans. However, no significant di#erences were observed in the ratio of those key amino acid residues thought to play a major role in modulating structural stability at subzero temperatures. For better comprehension of all of these features, it would also be helpful to compare each with a temperate species belonging to a phyletically related nototheniod family. All of the canonical residues required for proper folding of the VH domain to form the antigen-binding site were found. The high conservation of amino acid residues of both the entire FR4 region and the amino terminal part of CDR3 indicates that very similar JH segments are shared by the homologous sequences compared in (Fig. 2). Known and previously observed canonical structures could be identified in the sequences of the H1 and H2 loops (Chothia & Lesk, 1987; Chothia et al., 1989; Al-Lazikani et al., 1997). A positively charged residue (Arg) is present at position 94 and a negatively charged residue (Asp) is present at position 101 of the H3 loop suggesting that they form a double salt bridge and, consequently, that the loop has a ‘bulged’ conformation (Morea et al., 1998). In conclusion, the unusual features found for IgH chain from a coldadapted, species probably developed from a complex of events. It is proposed that di#erences found with respect to other fish species do not a#ect the overall activity of the molecule since hydrophobic core residues, which determine much of the stability of the final structure, are conserved. Indeed, amino acid insertions, possibly important for the internal mobility and flexibility of the molecule, might have a key role in its ability to function at low temperatures. We would like to thank Dr Laura Camardella for kindly providing tissues and Dr Ennio Cocca for his helpful advice on molecular cloning. Thanks are also due to Drs John Guardiola and Lars Pilström for their valuable comments on the manuscript. This research was supported by a grant from the Italian National Program for Antarctic Research.

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