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.     

Cystine-mediated oligomerization of the Atlantic salmon serum C-type lectin

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a C-type lectin that binds to bacteria including salmon pathogens. SSL has been shown to be oligomeric in salmon serum and it displays a stoichiometric band-laddering pattern when analyzed by SDS-PAGE under non-reducing conditions. In this study, a model was generated for SSL isoform 2 in silico in order to identify cysteines that are available to form intermolecular disulfide bonds facilitating oligomerization. Then, recombinant SSL was expressed in E. coli and mutants were produced at positions Cys72 and Cys149. The SSL preparations were purified by metal-affinity chromatography and shown to be functional by carbohydrate-affinity chromatography. The recombinant SSL formed oligomers, which were evident by non-reducing covalent cross-linking and non-reducing SDS-PAGE; however, the band patterns were different for the mutants, with the maximal and predominant multimer sizes distinct from the wild-type recombinant lectin. Further examination of oligomerization by size exclusion chromatography revealed a subunit number from 35 to at least 110 for the wild-type recombinant SSL and subunit numbers below 9 for each mutant SSL oligomer. Thus, both cysteines were found to contribute to oligomerization of SSL.     

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.     

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 a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5′-terminal untranslated region (UTR) of 60 bp and a 3′-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys⁵³, Cys¹²⁸, Cys¹⁴⁴, Cys¹⁵²) involved in the formation of disulfides bridges and the potential Ca²⁺/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca²⁺, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.     

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.     

Cloning and characterization of a novel animal lectin expressed in the rat sublingual gland.

We cloned a rat gene that is expressed primarily in the sublingual gland and named the predicted 503 amino-acid protein SLAMP (sublingual acinar membrane protein). SLAMP has 63% homology with human ERGIC-53-like protein, a member of the family of animal L-type lectins. Using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP, we examined the expression and localization of SLAMP in major rat organs and tissues. With both Northern and Western blot analyses, abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. With immunohistochemistry, SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. With immunoelectron microscopy, SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunner's glands in the duodenum. These results suggested that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells.     

Characterization of a recombinant C-type lectin, rCEL-IV, expressed in Escherichia coli cells using a synthetic gene

The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca2+-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for alpha-galactosides than for beta-galactosides, while other C. echinata lectins show preferential binding of beta-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses.     

Cloning and characterization of alpha-amylase from Atlantic salmon (Salmo salar L.)

Amylase has a lower activity in carnivorous fish species, particularly in Atlantic salmon. We report the first cloning of a salmonid alpha-amylase cDNA from Atlantic salmon, a major species in aquaculture. By amino acid alignment of several species, we identified a seven amino acid deletion in one of the large loops of the enzyme in relatively close proximity to the active site, that could impair substrate binding. We also found the signal peptide to be less hydrophobic compared to other species. This may affect import into ER during protein synthesis. Active site residues were shown to be conserved. Amylase mRNA expression was shown in pancreatic tissue, liver, and in the heart. Using blocked p-nitrophenyl-maltoheptaoside as a substrate, we measured a low amylase activity in Atlantic salmon intestinal content, which was about half of the activity measured in Atlantic cod, whereas activity measured in rainbow trout was fourteen times higher. Amylase activities in all three species showed similar degree of reduction in hydrolytic activity in a dose-response trial with a wheat amylase inhibitor preparation. This indicates similar specific activity per amylase molecule.     

Identification of a chicken CLEC-2 homologue, an activating C-type lectin expressed by thrombocytes

Receptors on natural killer (NK) cells are classified as C-type lectins or as Ig-like molecules, and many of them are encoded by two genomic clusters designated natural killer gene complex (NKC) and leukocyte receptor complex, respectively. Here, we describe the analysis of an NKC-encoded chicken C-type lectin, previously annotated as homologue to CD94 and NKG2 and thus designated chicken CD94/NKG2. To further elucidate its potential function on NK cells, we produced a specific mab by immunizing with stably transfected HEK293 cells expressing this lectin. Staining of various chicken tissues revealed minimal reactivity with bursal, or thymus cells. In peripheral blood mononuclear cell and spleen, however, the mab reacted with virtually all thrombocytes, whereas most NK cells in organs such as embryonic spleen, lung and intestine were found to be negative. These findings indicate that the gene may not resemble CD94/NKG2, but rather a CLEC-2 homologue, a claim further supported by sequence features such as an additional extracellular cysteine residue and the presence of a cytoplasmic motif known as a hem immunoreceptor tyrosine-based activation motif, found in C-type lectins such as Dectin-1, CLEC-2, but not CD94/NKG2. The biochemical analyses demonstrated that CLEC-2 is present on the cell surface as heavily glycosylated homodimer, which upon mab crosslinking induced thrombocyte activation, as measured by CD107 expression. These analyses reveal that the chicken NKC may not encode NK cell receptor genes, in particular not CD94 or NKG2 genes, and identifies a chicken CLEC-2 homologue.     

Isolation, purification, and partial characterization of a lectin from chinook salmon ova

Chinook salmon (Oncorhynchus tshawytscha) ova contain a lectin that agglutinated human type B and rabbit erythrocytes and was specifically inhibited by the monosaccharides d-galactose and l-rhamnose. The lectin purified from homogenates of the ova by affinity chromatography on agarose possessed a pI of 4.5 in isoelectric focusing studies. The purified lectin inhibited the growth of four bacterial fish pathogens.     

Gene expression and molecular characterization of a novel C-type lectin,encapsulation promoting lectin (EPL), in the rice armyworm,Mythimna separata

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated “encapsulation promoting lectin (EPL)” regulates cellular immunity by a novel immune target size-recognition mechanism.     

Tetranectin, a trimeric plasminogen-binding C-type lectin.

Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization.     

Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene

CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.     

Structural and biological characterization of Nattectin, a new C-type lectin from the venomous fish Thalassophryne nattereri

Lectins are glycan-binding receptors that recognize glycan epitopes on foreign pathogens and in the host systems. They can be involved in functions that include innate immunity, development, immune regulation and homeostasis. Several lectins have been purified and characterized from fish species. In this work, using cation-exchange chromatography, a galactose-specific lectin belonging to the family of C-type lectins was isolated from the venom of the Brazilian venomous fish Thalassophryne nattereri. Nattectin is a basic, non-glycosilated, 15 kDa monomeric protein. It exhibits hemagglutination activity that is independent of Ca²⁺. We also demonstrated a lectin activity for Nattectin in the innate immune system, especially in neutrophil mobilization in mice, indicating that marine organisms are source of immunomodulator agents.     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.