Broad phylogenetic expression of heavy-chain determinants detected by rabbit antisera to VHa allotypes.

Immunoglobulin molecules from diverse vertebrate species were examined, using an enzyme-linked immunosorbent assay (ELISA), for the expression of determinants detectable by rabbit antisera to VHa allotypes. The data indicate that immunoglobulins of elasmobranchs, teleosts, amphibians and birds express determinants cross-reactive with those specified by the a1, a2 and a3 alleles in the domestic rabbit. We localize VHa cross-reactive specificity to the denatured heavy chain of a primitive vertebrate, the Galapagos shark (Carcharhinus galapagensis). Furthermore, the N-terminal amino acid sequence of the shark heavy chain shows significant homology with rabbit heavy chains of known VHa type at positions where allotype-correlated differences have been implicated. VHa-related determinants are shared by immunoglobulins of a wide range of vertebrates from sharks to man and thus seem to be epitopes which have been conserved during vertebrate evolution. The determinants detected on immunoglobulins of lower vertebrates by rabbit anti-VHa allotype sera most probably are VH-subgroup rather than allotypic markers. Their distribution demonstrates a strong phylogenetic conservation of VH-regions.

Beta 2 microglobulin-like polypeptide of the goldfish, Carassius auratus.

Antisera to human beta 2 microglobulin (beta 2M) detected a plasma membrane molecule on goldfish (Carassius auratus) cells in immunofluorescence. A goldfish molecule detected by radioimmunoassay (RIA) co-eluted with human beta 2M on gel filtration. By affinity chromatography on immobilized antibody to human beta 2M, a molecule was purified (from extracts of goldfish) that showed, on SDS-polyacrylamide gel electrophoresis, a mobility similar to that of human beta 2M (apparent Mr 12,800 +/- 500).

Partial characterization of immunoglobulin light chains of carcharhine sharks: evidence for phylogenetic conservation of variable region and divergence of constant region structure.

Isolated light chains of IgM-type immunoglobulins of carcharhine sharks were analyzed by serological and biochemical means. When analyzed by isoelectric focusing analysis, light chains of the tiger shark (Galecerdo cuvieri), the galapagos shark (Carcharhinus galapagensis) and the sandbar shark (Carcharhinus plumbeus) showed a broad, but patterned, spectrum of bands ranging from pI 5.0 to 7.7 in which discrete families were observed. Serologically, light chains of the galapagos shark cross-reacted with rabbit antibodies against mouse immunoglobulin and a synthetic peptide corresponding to the J segment of T cell receptor beta chain. The latter cross-reaction is shared among light chains and T cell receptors. Although there was considerable heterogeneity in isoelectric focusing analysis, the light chains were homogeneous on the basis of apparent mass (23 kDa) and those of tiger shark and galapagos shark had relatively homogeneous dominant N-terminal sequences representing the first framework. The N-terminal sequences of these two shark light chains, were strongly homologous to one another and showed 75% identity to certain V kappa sequences of man and dog. Homology was also shown to V lambda sequences, but the degree of identity was approximately 50%. Following cleavage of the tiger shark light chain with o-iodosobenzoic acid which cleaves at tryptophanyl residues, a constant region peptide was isolated by gel filtration. It was possible to identify the homolog of this peptide within the constant regions of mammalian kappa and lambda chain, but the relationship to C kappa chain was stronger. The degree of identity among the corresponding C region peptides of mammalian, avian and elasmobranch species was much less than that observed for the framework 1 sequence of the light chain variable region. These data support the concept that variable and J region sequence have been conserved in the evolution of placoderm-derived vertebrates, but that constant regions show much greater phylogenetic variation.

Conservation among the immunoglobulins of carcharhine sharks and phylogenetic conservation of variable region determinants.

To study primitive vertebrate recognition molecules we have purified the immunoglobulins of three species of carcharhine sharks. The tiger (Galeocerdo cuvieri), the sandbar (Carcharhinus plumbeus) and the galapagos sharks (Carcharhinus galapagensis) possess 18S high molecular weight and 7S low molecular weight immunoglobulin forms. Both within and between species these forms closely resemble each other in polypeptide chain composition, heavy chain mass, carbohydrate content, amino acid composition, antigenic character and amino terminal sequence. These carcharhine species are separated by at least 30 million years of evolutionary time and it is remarkable that so little diversification of immunoglobulin structure has occurred during their evolution. By several criteria carcharhine immunoglobulins most closely resemble mammalian IgM. Studies of serological cross-reactions indicate that some immunoglobulin determinants have been conserved over a broad phylogenetic range of vertebrate classes; most notably JH and VHa-related markers are shared between forms as diverse as sharks and mammals.

The immunoglobulins of carcharhine sharks: a comparison of serological and biochemical properties.

The immunoglobulins of three carcharhine sharks were isolated from serum by means of salt precipitation and gel chromatography. The Galapagos shark (Carcharhinus galapagensis), the sandbar shark (Carcharhinus plumbeus) and the tiger shark (Galeocerdo cuvieri) each contained high molecular weight (18S) and low molecular weight (7S) IgM-like molecules as the major serum immunoglobulins. Both within and between species 18S and 7S immunoglobulins closely resemble each other in antigenic character, polypeptide chain composition, chain mass, amino acid composition, carbohydrate content and amino-terminal sequence. These results suggest that the immunoglobulins of carcharhine sharks have undergone little structural divergence during their evolution.

Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide.

We used affinity purified antibodies produced against a synthetic peptide sequence corresponding to the entire J beta of a human T cell receptor gene to screen sera of man, mouse and other vertebrates to determine the presence of cross-reactive molecules. Little evidence for free alpha/beta heterodimers was found, but the antibody reacted with light chains of many vertebrate species, including characterized myeloma proteins of man and mouse. Some vertebrate orders, notably Aves, lacked polypeptide chains cross-reactive with J beta, but detectable determinants occurred in primitive vertebrates such as the galapagos shark (Carcharhinus galapagensis). In addition to the strong cross-reaction with purified light chains, human heavy chains reacted weakly with the antibody. The cross-reaction correlated with the sequence of the denatured immunoglobulins and was inhibitable with free peptide. These results establish the similarity of T cell receptor beta chains to immunoglobulin chains and support the conclusion that J region sequences were conserved, not only within mammalian immunoglobulins and T cell receptors, but in vertebrate evolution.