Serum carbohydrate-binding IgM are present in Vietnamese striped catfish (Pangasianodon hypophthalmus) but not in North African catfish (Clarias gariepinus)

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 Ca2+ 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 Ca2+ 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.

► Pangasianodon hypophthalmus serum contains carbohydrate-binding IgMs.
► Carbohydrate-binding activity is dependent on calcium.
► The IgMs are tetramers that consist of non-covalently linked monomers and halfmers.
► Monomers and halfmers exist as intra-chain redox forms.
► Serum carbohydrate-binding IgMs agglutinate fish pathogenic bacteria.