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.
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada.
David M
DM
Hudson
Neil R
NR
Mattatall
Elke
E
Uribe
Robert C
RC
Richards
Huansheng
H
Gong
K Vanya
KV
Ewart
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003553:Cystine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029941:Fish Proteins / Q000097:blood* / Q000737:chemistry* / Q000235:genetics; D007113:Immunity, Innate; D037181:Lectins, C-Type / Q000097:blood* / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020317:Salmo salar / Q000097:blood* / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein
D003432:Cross-Linking Reagents; D017931:DNA Primers; D029941:Fish Proteins; D037181:Lectins, C-Type; D021122:Protein Subunits; D011994:Recombinant Proteins; D003553:Cystine