P.E.E. Todd

The immunogenicity and antigenicity of proteins

Abstract The exploration of antigenic regions on the surface of proteins and viruses has become easier with the advent of monoclonal antibodies and rapid methods peptide synthesis. For small proteins of known sequence and conformation - myoglobin, cytochrome c, insulin and lysozyme - the antigenic sites may be described as surface domains made up from amino acid side chains which may be distant in sequence but close in space. Such domains are probably overlapping and cover most of the protein surface.

On topographic antigenic determinants in myoglobins

Abstract A study of the antigenic cross-reactivities of beef, sheep and pig myoglobins is reported, in which attention is focused on a single determinant. The amino acid sequence of this determinant (residues 15–22) is identical in beef and sheep myoglobins and only six other residues differ in the rest of their 153 residues. An anti-beef myoglobin antibody fraction which binds specifically to beef myoglobin peptide (1–55) is shown to contain some antibodies which bind equally well to beef and sheep myoglobins and other antibodies which bind more strongly to the beef protein. The evidence from this and several other sources is discussed in the light of current hypotheses on the topographic nature of antigenic determinants.

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.

Cross-reactivity between mammalian myoglobins: Linear vs spatial antigenic determinants

Abstract Cross-reactivities between myoglobins from sperm whale, horse, pig and beef have been measured by radioimmunoassay procedures. Taking into account the high degree of homology of the amino acid sequences in the regions believed to be antigenic in the four proteins, the effect of amino acid substitutions outside these regions is unexpectedly large. The spatial character of antigenic domains in proteins is discussed in the light of these findings.

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.

Original antigenic sin: experiments with a defined antigen.

Abstract The phenomenon referred to as ‘Original Antigenic Sin’ is the production of antibodies to a specific antigen elicited by a boosting injection of a related antigen. We have investigated this phenomenon using myoglobins of known amino acid sequence and structure. Our results indicated that in order to elicit an ‘Original Antigenic Sin’ response the boosting myoglobin should differ by less than 33–42% in overall sequence from the priming myoglobin. The antibodies in the antisera from rabbits primed with beef myoglobin and boosted with a different myoglobin from a different species are predominantly and perhaps exclusively directed towards antigenic regions common to the priming and boosting myoglobins but have a higher affinity for the priming myoglobin.

Purification and properties of adrenal acid proteinase.

Abstract The proteinase activities at pH 3.5 (haemoglobin substrate) of extracts of a number of bovine tissues, including endocrine glands, have been compared. All endocrine extracts tested were highly active at this pH. Extracts of thyroid and of adrenal glands, when tested for proteinase activity (haemoglobin substrate) over a range of pH values, showed two pH optima, one in the acid and one in the alkaline range, thus resembling extracts of pituitary glands. A proteinase optimally active between pH 3.5 and pH 4.0 has been purified approx. 70-fold from extracts of bovine adrenal glands and some of its properties are reported. The enzyme had no action on any of the synthetic peptide substrates tested.

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 hosts 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 hosts 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.

Hydrolysis of the A and B chains of oxidised insulin by adrenal acid proteinase

Abstract The hydrolysis of the A and B chains of oxidised insulin by adrenal acid proteinase has been investigated qualitatively and quantitatively. It was found that the bonds hydrolysed most readily were TyrLeu, LeuTyr, PhePhe and PheTyr. The other susceptible bonds were those formed from glutamyl, alanyl, leucyl, valyl or asparaginyl residues. This limited hydrolysis by the enzyme was supported by experiments using the following substrates: ribonuclease (EC 2.7.716), unoxidised insulin, oxidised ribonuclease and some synthetic peptides and peptide derivatives. From the qualitative results and a comparison betwee hydrolysis at pH 3.6 and pH 5.3 it seems that the presence of ionised groups inhibits the action of the enzyme.

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.