The disulfide bonding pattern in ficolin multimers

Ficolin is a plasma lectin, consisting of a short N-terminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin alpha in which the N-terminal cysteines were substituted by serines (Cys4, Cys24, and Cys4/Cys24). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfide-linked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers. The Cys4 mutant also formed 12-mers, but Cys24 and Cys4/Cys24 mutants formed only trimers. This means that protein interfaces containing Cys4 are stable as noncovalent protein-protein interactions and do not require disulfides, whereas those containing Cys24-Cys24 require the disulfides for stability. Proteins were also analyzed by nonreducing SDS-PAGE to show the covalent structure under denaturing conditions. Wild-type ficolin was covalently linked into 12-mers, whereas elimination of either Cys4 or Cys24 gave dimers and monomers. We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers.     

Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl₂ induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.     

Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition

A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria. Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host. In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting. A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl alpha-D-mannopyranoside to be additional ligands of this lectin. The SSL in serum detected by Western blotting was shown to form a complex oligomer. These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose. The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces. No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody. Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species. It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species.     

Cloning and characterization of the Atlantic salmon serum lectin,a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.     

Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.

Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.     

Cloning and characterization of the Atlantic salmon serum lectin,a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.     

Role of disulfide bonds in biologic activity of human interleukin-6.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.     

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.     

A Rainbow Trout Lectin with Multimeric Structure

A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, l-fucose, maltose and α-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and d-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH₂-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein “ladderlectin.”     

In vivo formation and stability of engineered disulfide bonds in subtilisin

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.     

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.     

Dissecting the individual contribution of conserved cysteines to the redox regulation of RubisCO

Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.     

Structural roles of subunit cysteines in the folding and assembly of the DNA packaging machine (portal) of bacteriophage P22

The DNA packaging machine (portal assembly) of bacteriophage P22 is constructed from 12 copies of a multidomain 725-residue subunit comprising a complex alpha/beta fold. The portal subunit contains four cysteines (Cys 153, Cys 173, Cys 283, and Cys 516), which produce distinctive Raman markers in the spectral interval 2500-2600 cm(-1) originating from S-H bond-stretching vibrations diagnostic of S-H...X hydrogen-bonding interactions. The Raman spectrum is unique in the capability to characterize cysteine sulfhydryl interactions in proteins and shows that portal cysteine environments are significantly altered by assembly (Rodriguez-Casado et al. (2001) Biochemistry 40, 13583-13591). We have employed site-directed mutagenesis, size-exclusion chromatography, and Raman difference spectroscopy to characterize the roles of portal cysteines in subunit folding and dodecamer assembly. The stability of the portal monomer is severely reduced by a Cys --> Ser point mutation introduced at either residue 173 or 516. In the case of C516S, the destabilized monomer still forms portal rings, as visualized by negative-stain electron microscopy, whereas portal ring formation cannot be detected for C173S, which forms aberrant aggregates. The C283S mutant is a hyperstable monomer that is defective in portal ring formation. Interestingly, Cys 283 is suggested by secondary structure homology with the phi29 portal to be within a domain involved in DNA translocation. Conversely, the phenotype of the C153S mutant is close to that of the wild-type protein, implying that the sulfhydryl moiety of Cys 153 is not essential to formation of the native subunit fold and productive assembly dynamics. The present results demonstrate that cysteines of the P22 portal protein span a wide range of sulfhydryl hydrogen-bonding strengths in the wild-type assembly, that three of the four sulfhydryls play key roles in portal protein stability and assembly kinetics, and that substitution of a mutant seryl interaction (O-H...X) for a wild-type cysteinyl interaction (S-H...X) can either stabilize or destabilize the native fold depending upon sequence context.     

Characterization of the oligomer structure of recombinant human mannan-binding lectin

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization.     

Identification by site-directed mutagenesis and chemical modification of three vicinal cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter

The proximity of the Cys residues present in the mitochondrial rat carnitine/acylcarnitine carrier (CAC) primary structure was studied by using site-directed mutagenesis in combination with chemical modification. CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli and reconstituted into liposomes. The effect of SH oxidizing, cross-linking, and coordinating reagents was evaluated on the carnitine/carnitine exchange catalyzed by the recombinant reconstituted CAC proteins. All the tested reagents efficiently inhibited the wild-type CAC. The inhibitory effect of diamide, Cu(2+)-phenanthroline, or phenylarsine oxide was largely reduced or abolished by the double substitutions C136S/C155S, C58S/C136S, and C58S/C155S. The decrease in sensitivity to these reagents was much lower in double mutants in which Cys(23) was substituted with Cys(136) or Cys(155). No decrease in inhibition was found when Cys(89) and/or Cys(283) were replaced with Ser. Sb(3+), which coordinates three cysteines, inhibited only the Cys replacement mutants containing cysteines 58, 136, and 155 of the six native cysteines. In addition, the mutant C23S/C89S/C155S/C283S, in which double tandem fXa recognition sites were inserted in positions 65-72, i.e. between Cys(58) and Cys(136), was not cleaved into two fragments by fXa protease after treatment with diamide. These results are interpreted in light of the homology model of CAC based on the available x-ray structure of the ADP/ATP carrier. They indicate that Cys(58), Cys(136), and Cys(155) become close in the tertiary structure of the CAC during its catalytic cycle.     

Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin.

The roles of selected amino acid residues of human 14-kDa beta-galactoside-binding lectin were studied by site-directed mutagenesis. Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate beta-galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted. All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin. In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions. It retained asialofetuin binding activity for over a week in the absence of beta-mercaptoethanol, while the wild-type lectin lost it within a day. This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin. Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose. Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation. Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential.     

Ligand and pathogen specificity of the Atlantic salmon serum C-type lectin

Background

An Atlantic salmon (Salmo salar) C-type lectin (SSL) binds to mannose and related sugars as well as to the surface of Aeromonas salmonicida. To characterize this lectin as a pathogen recognition receptor in salmon, aspects of its interaction with molecules and with intact pathogens were investigated.

Methods

SSL was isolated using whole-yeast-affinity and mannan-affinity chromatography. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays. Microbial binding specificity of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand specificity of SSL was examined using glycan array analysis and frontal affinity chromatography.

Results

SSL showed binding to bacteria and yeast including, Pseudomonas fluorescens, A. salmonicida, A. hydrophila, Pichia pastoris, and Saccharomyces cerevisiae, but there was no detectable binding to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against Escherichia coli, Bacillus subtilis, S. cerevisiae, or A. salmonicida, but it was found to agglutinate E. coli. The major surface molecule of A. salmonicida recognized by SSL was shown to be LPS and not the A-layer protein. LPS binding was mannose-inhibitable. Glycans containing N-acetylglucosamine were shown to be predominant ligands.

Conclusion

SSL has a distinct ligand preference while allowing recognition of a wide variety of related carbohydrate structures.

General Significance

SSL is likely to function as a wide-spectrum pattern recognition protein.     

The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state

MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus- intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C- terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.      

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai

A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.     

Characterization of highly concentrated serum lectins in spotted halibut Verasper variegatus

Mannose-binding lectins were purified from flatfish spotted halibut (Verasper variegatus) serum. These lectins, which we named VVL (Verasper variegatus lectin)-alpha (approximately 33 kDa) and VVL-beta (approximately 30 kDa) (VVLs), under non-reducing SDS-PAGE, were surprisingly highly concentrated in serum (1.92+/-0.55 mg/ml; n=5), compared with other serum lectins. Both VVLs are heterodimers comprised of 2 types of subunit via inter-subunit disulfide bonds, and one subunit of VVL-alpha has a N-linked sugar chain. Based on N-terminal amino acid sequences, the nucleotide sequences of one subunit of VVL cDNAs were determined by 3'- and 5'-rapid amplification of the cDNA ends. The full-length VVL subunit cDNAs contained 489 bp, encoding an open reading frame of 163 amino acids. We found that VVLs bind to an approximately 8 kDa ciliary surface glycoprotein (a putative agglutination/immobilization antigen that we reported previously) of the fish parasite Neobenedenia girellae, and agglutinate this parasite in vitro.