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Atlantic salmon (Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CmuA and CmuB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout (Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CmuA and CmuB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CmuB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cmu subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.

The assembly of fully disulfide bonded trout Immunoglobulin M (IgM) was modeled heuristically. The basic mechanism was assumed to be the sequential formation of single disulfide bonds (DSB), linking two monomeric subunits. Biochemical details of the mechanisms of bond formation were not considered. The progression of bonding within each tetramer was described entirely in terms of the rates of bond formation. It was further assumed that the sequence of DSB formation from non-covalently associated tetramers to fully bonded tetramers progressed by the formation of one bond at a time. The experimentally observed ratios of covalently-linked subunits within the tetramers were compared with the values predicted by the models. The results of these analyses suggest that the final stages of trout Ig assembly (the DSB formation between monomeric subunits) must occur in intracellular compartments late in the secretory process itself, thereby yielding incompletely crosslinked tetramers. Further, it would appear that the rate of disulfide bond formation within a tetramer may be accelerated by the presence in that tetramer, of previously incurred, intermonomeric DSBs., (Copyright 1998 Academic Press.)

A complete cDNA clone encoding secreted IgM molecules was isolated from a spleen cDNA library of rainbow trout (Oncorhynchus mykiss). It encodes the leader peptide, VH, DH, JH, and the whole constant region of the secreted IgM molecule. We also studied the copy number of the IgM gene and an unusual RNA splicing mechanism which generates membrane IgM lacking the CH4 domain in rainbow trout. Amino acid sequence comparison of IgM protein sequences from this fish and other vertebrates indicates that some domains of IgM have evolved at a relatively constant rate. The evolution of salmonid fish including divergence time is discussed.

Monoclonal antibodies to rainbow trout (Oncorhynchus mykiss) IgM were prepared and characterized for use in immunoassays. Antibodies produced by the five clones reacted with the heavy chain of the immunoglobulin. No indication of different heavy chain isotype specificity was observed for the MAbs. One clone discerned IgM from rainbow trout while the other four clones cross-reacted with IgM from Atlantic salmon (Salmo salar) and from brown trout (Salmo trutta). The monoclonal antibodies identified a B-cell like lymphocyte population that contributed to approximately 45% of the blood leucocytes in rainbow trout but was absent in the thymus. The proportion of Ig+ cells was higher in blood lymphocyte cultures stimulated with lipopolysaccharide than in nonstimulated cultures or in cultures stimulated with Concanavalin A. Applied in an ELISA for measuring humoral antibodies to Vibrio anguillarum in trout, the monoclonal anti-rainbow trout IgM antibodies discriminated seropositive fish from control fish more efficiently than did polyclonal rabbit antitrout IgM antibodies.

A monoclonal antibody to trout serum IgM was tested by immunofluorescence analysis with lymphocytes from thymus, spleen and head kidney. By visual examination, the antibody reacted with only a subpopulation of lymphocytes. The mean values +/- SE for positive cells were 5.2 +/- 2.3% in the thymus, 30.3 +/- 7.9% in the spleen and 12.4 +/- 3.0% in the head kidney. Flow cytofluorometric analysis revealed evidence of heterogeneity by size among the membrane IgM-positive cells of the head kidney and spleen. Depletion of head kidney cells positive for surface IgM by an immune affinity adherence technique of panning, using monoclonal anti-IgM, significantly reduced the mitogenic response to lipopolysaccharide but not to concanavalin A. It is suggested that this information supports the existence of distinct subpopulations of fish lymphocytes that may be homologous in certain respects to mammalian T and B type cells.

Monomeric and tetrameric IgM anti-haptin antibodies isolated from the sera of rainbow trout (S. gairdnerii) by immunoaffinity chromatography were compared both immunochemically and with regard to their functional abilities to lyse haptenated trout erythrocytes in the presence of trout complement. The two populations had similar binding affinities for hapten and apparently identical L chains, but differed in H chain peptide maps and immunoreactivity with rabbit anti-trout H chain serum. These differences could not be attributed to J-chain. The abilities of the two antibody subpopulations to activate C to lyse haptenated trout erythrocytes also differed dramatically. Such functional differences are not simply explained by the greater avidity of the tetrameric form since preliminary studies show that the monomeric form of trout IgM activates C via an alternative pathway mechanism while the tetrameric form activates both classical and alternative pathway mechanisms. Results suggest divergent evolution of antibody structures involved in the familiar effector functions (C activation, transport, etc.).