Antibody response to the C-terminal peptide sequence in beef myoglobin.

The nature of the surface determinants on the beef myoglobin molecule which direct the distinctive antibody response of sheep, have been further defined. Antisera raised to beef myoglobin in sheep, rabbits and mice have been compared for their ability to recognize the synthetic C-terminal beef myoglobin peptide (140-153), which contains four of the six amino acid substitutions between sheep and beef myoglobins. The antibodies raised in rabbits, directed to the entire surface of beef myoglobin, contain only a minor population directed to the C-terminal sequence: in mice, even fewer antibodies are specific for this sequence. In sheep, however, antibodies to beef myoglobin appear to be directed almost exclusively to topographic domains which include the (140-153) sequence. This specificity is most apparent in "early" antisera in which all antibodies display equal avidity for "native" beef myoglobin and the peptide; further immunization produces antibodies which recognize a larger overlapping set of domains and only 20% of these antibodies have effective avidity for the (140-153) peptide. The antibodies to beef myoglobin raised in sheep comprise two discrete populations. One ("common") population is directed to regions of similarity between the beef and sheep myoglobin molecules, in which the region represented by the C-terminal peptide of beef myoglobin is less important in defining the antibody-binding site and/or affinity, while still being directly involved in the topographic determinant. The other ("non-common") appears to be directed almost exclusively to the C-terminal sequence (140-153) of beef myoglobin. The findings are discussed in relation to our previous findings on the effect of the host species on the nature of the antibody response and in relation to views on the possibility of direct vs indirect effects of evolutionary amino acid substitution on immuno-cross-reactivity among homologous proteins.

Two large immunogenic and antigenic myoglobin peptides and the effects of cyclisation.

Peptides corresponding to sequences (72-88) and (26-54) of beef myoglobin have been synthesised in their open-chain and cyclised forms (using a disulphide bridge) and tested for their antigenicity and immunogenicity. Antibodies raised to beef myoglobin bound to both peptides but more strongly to the 29-residue than to the 17-residue peptide. Cyclisation increased the antigenicity of the larger peptide. In this form the peptide competed much more strongly than in the uncyclised form for specific antibodies to beef myoglobin. The peptides are immunogenic in mice without being coupled to a protein carrier and produce antibodies which bind to beef myoglobin. Peptide (26-54) is the more immunogenic in producing a larger antibody titre to the parent myoglobin and cyclisation again enhances this property. The findings lend weight to the view that longer peptide sequences might be expected to favour the folded state, therefore binding more strongly to antibodies raised to the native protein and eliciting a population of antibodies which contain a larger proportion specific for that conformation. Cyclisation enhances antigenicity and immunogenicity presumably by decreasing the number of degrees of conformational freedom of a peptide without excluding native-like conformations.

Original antigenic sin and the production of autoantibodies.

Autoantibodies to sheep myoglobin have been raised by priming sheep with beef myoglobin and boosting with sheep myoglobin. The autoantibodies appear to be a subset of those produced when beef myoglobin is used for both priming and boosting. This subset of antibodies is presumably directed to the surface regions which are common to both myoglobins. The antibodies which bind to sheep myoglobin in the 2 types of antisera differ. Those elicited by boosting with beef myoglobin bind better to beef myoglobin than to sheep myoglobin, while those obtained by boosting with sheep myoglobin bind with equal avidity to the 2 myoglobins. It would seem therefore that the boosting immunogen determines which fraction of antibodies is selected from the antibody repertoire established by the priming immunogen. Our results also show that tolerance at the T-cell level can be circumvented by exposing the immune system to a protein closely related to a homologous self protein.

Antibody response to myoglobins: effect of host species.

Using both direct and competitive binding studies it is demonstrated that antibodies to beef myoglobin raised in sheep are able to distinguish between beef and sheep myoglobins although these two proteins differ by only six of the 153 amino acid residues. By contrast, antibodies to beef myoglobin raised in rabbits, dogs and chickens bind almost equally well to beef and sheep myoglobins. It is also shown that antibodies to beef myoglobin raised in sheep have a lower avidity for beef myoglobin than do antibodies raised in more distantly related species. Furthermore, only 50% of the specific anti-beef myoglobin antibodies isolated from sheep antisera will bind to sheep myoglobins whereas 100% of the specific antibodies isolated from the antisera of the other immunised species will bind to sheep myoglobin. It is suggested that antibodies to beef myoglobin are raised to those surface regions which are topographically altered as a result of sequence differences from the host's own myoglobin. When the host animal is evolutionarily distant these sequence differences are considerable and antibodies are raised to the entire surface of the molecule. However, when the host's myoglobin is very similar in sequence to beef myoglobin (as is the case when using sheep as the host animal) antibodies are made only to surface regions affected by the sequence differences. Some of these antibodies--those to the regions of greatest difference--will bind weakly if at all to sheep myoglobin, while those directed to areas of lesser difference will bind well to sheep myoglobin.

The antigenicity of myoglobin-related peptides synthesised on polyacrylamide and polystyrene resin supports.

Polyacrylamide resins [Atherton et al., Bioorg. Chem. 8, 351-370 (1979)] have been found suitable for solid-phase radioimmunoassay of peptides synthesised on the same supports; they are sufficiently stable during side-chain deprotection and swell sufficiently in aq. media to admit antibody molecules to the sites of peptide attachment. A re-examination of five synthetic peptide sequences corresponding to (15-21), (56-62), (94-99), (113-119) and (145-151) of beef myoglobin analogous to those delineated by Atassi [Immunochemistry 12, 423-438 (1975)] for sperm whale myoglobin shows that they all bind anti-beef myoglobin antibodies raised in rabbits, with binding capacities in the order V = III greater than IV greater than I = II. The resin-bound peptide (72-88) binds such antibodies even more extensively, as do certain sequential variants of peptide V. Other peptides, bound to polyacrylamide or polystyrene resins but unrelated to any of the five sequences and varying in size and amino acid composition and sequence were also tested with various antisera. It was concluded that the antibody binding properties of the 30 or so small peptides (two-seven residues) are dominated by their cationic and/or hydrophobic properties. In small peptides, therefore, antibody binding can be safely interpreted only in terms of general structural properties but not in terms of biological specificity. The latter property becomes assessable only with peptides representing larger areas of antigenic protein surfaces.

Antigenic specificity of monoclonal antibodies to human myoglobin.

Two monoclonal antibodies directed against different sites of the human myoglobin molecule have been tested for their cross-reactivities against several myoglobins including seven from mammalian species. The relation between their cross-reactivities and their amino acid sequences had led to a possible localization of two antigenic domains in human myoglobin. Each domain includes residues previously considered not to be directly involved in the antigenic structure of myoglobin. Unlike polyclonal serum antibodies, monoclonal hybridoma antibodies directed to a native protein often fail to bind to supposedly antigenic protein fragments. This is explicable in terms of the concept of antigenic domains. Such domains are numerous and overlapping, each comprising a number of contributory amino acid side chains which need not necessarily include continuous sequences of amino acids and which need not exhibit measurable antigenicity in isolation from the rest of the domain.

Comparative aspects of aminotransferases in the rat, pigeon and rainbow trout.

1. The activities of aminotransferases catalysing the transfer of amino groups from aspartate, alanine and leucine to 2-oxoglutarate in different tissues of the rat, pigeon and trout have been determined. 2. Alanine-2-oxoglutarate aminotransferase was high in the liver of the rat and trout and low in that of the pigeon. 3. Aspartate-2-oxoglutarate aminotransferase was usually the dominant aminotransferase in all tissues and was highest in oxidative tissues where the TCA cycle is active. Its activity in the various livers is not correlated with the function of aspartate in nitrogen excretion. 4. The activity of aspartate-2-oxoglutarate aminotransferase in oxidative tissues argues that aspartate in conjunction with this enzyme serves as a buffer of oxaloacetate to keep the TCA cycle running and/or to mediate the transfer of reducing equivalents across mitochondrial membranes.