Comparative analysis of IgM sub-variants in salmonid fish and identification of a residue in μ3 which is essential for MAb4C10 reactivity

Abstract

In rainbow trout (Oncorhynchus mykiss) it has been shown that high affinity IgM antibodies have a higher degree of disulfide polymerization and a longer half life time. In the present study, distinct IgM sub-variants related to ancestral tetraploidy in salmonid fish were analyzed to reveal possible characteristic differences between these. A monoclonal antibody (MAb4C10) which distinguishes between IgM-A and IgM-B in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) was further characterized. It was shown that substitution of a proline located in the loop between the B and C beta strands of the third constant domain (μ3) of salmon μA eliminated MAb4C10 reactivity. Accordingly, the reverse substitution in salmon μB restored MAb4C10 reactivity. Molecular cloning of μ cDNA from arctic char (Salvelinus alpinus) revealed two sub-variants (μA-1 and μA-2), i.e. a similar situation as in Atlantic salmon and brown trout. However, arctic char IgM eluted in one peak by anion exchange chromatography, in contrast to salmon and brown trout IgM that are eluted in two peaks. The only characteristic residue of salmon and brown trout μB is an additional cysteine in the C-terminal part of μ4. Most likely, this cysteine is involved in inter-chain disulfide bonding and influences the elution profiles of IgM-A and IgM-B on anion exchange chromatography. Neither of the μ sub-variants in arctic char have the additional cysteine, and char IgM, as well as salmon and brown trout IgM-A, showed a lower degree of inter-chain disulfide bonding than IgM-B when subjected to denaturation and gel electrophoresis under non-reducing conditions. Hybrids of char/salmon expressed μA-1, μA-2, μA and μB, indicating that there are two paralogous Ig heavy chain gene complexes in the haploid genome of char, like in Atlantic salmon. A comparison of salmonid μ sequences is presented, including representatives of Salmoninae (trout, salmon and char), Thymallinae (grayling) and Coregoninae (whitefish).