title: Serum carbohydrate-binding IgM are present in Vietnamese striped catfish (Pangasianodon hypophthalmus) but not in North African catfish (Clarias gariepinus).
abstract: Pangasianodon hypophthalmus serum was fractionated by affinity chromatography on 12 different Sepharose-carbohydrate columns and proteins eluted by the corresponding sugar. Binding to the affinity matrices is dependent on Ca(2+) ions. Upon gel filtration using Superose-12, essentially one fraction was obtained, eluting as a protein with a molecular mass of about 900 kDa. SDS-PAGE in reducing conditions revealed the presence of large (72 kDa) subunits (H-chains) and one up to three small (24, 26 and/or 28-29 kDa) subunits (L-chains). The isolated proteins were shown to be IgM since they bind monoclonal anti-P. hypophthalmus IgM antibodies. Rabbit polyclonal anti-galactose-binding IgM only cross-react with some sugar-binding IgM. The H-chains of the anti-carbohydrate IgM are glycosylated. Circular dichroism studies revealed that the IgMs have an "all-β" type of structure, and that Ca(2+) ions, though essential for carbohydrate-binding activity, are not required for the structural integrity of the molecules. In non-reducing SDS-PAGE, only monomers and halfmers were obtained, showing that there are no disulfide bonds linking the monomers, and that a disulfide bond connecting both H-chains within one monomer is only present in 45% of the molecules. Both the monomers and the halfmers display molecular mass heterogeneity which is indicative for redox forms at the level of the intradomain disulfide bonds. The native carbohydrate-binding IgMs agglutinate erythrocytes from different animals, as well as fish pathogenic bacteria. Similar proteins could not be isolated from another catfish, Clarias gariepinus.
authors.affiliation: Laboratory of Protein Chemistry, Vrije Universiteit Brussel, Brussel, Belgium.
authors.forename: Duong Thi Huong
authors.initials: DT
authors.lastname: Giang
authors.forename: Edilbert
authors.initials: E
authors.lastname: Van Driessche
authors.forename: Sonia
authors.initials: S
authors.lastname: Beeckmans
mesh_terms: D000372:Agglutination Tests / Q000662:veterinary; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002118:Calcium / Q000276:immunology; D002397:Catfishes / Q000097:blood / Q000276:immunology*; D002846:Chromatography, Affinity / Q000662:veterinary; D002850:Chromatography, Gel / Q000662:veterinary; D002942:Circular Dichroism / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D007143:Immunoglobulin Heavy Chains / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D007075:Immunoglobulin M / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D037102:Lectins / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet / Q000662:veterinary
chemical_list: D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D037102:Lectins; D002118:Calcium