The Functional Dissection of an Antigen Molecule: Specificity of Humoral and Cellular Immune Responses to Glucagon

Abstract

Bovine glucagon, a polypeptide of 29 amino acids, was immunogenic in guinea pigs. The immunologic determinants of glucagon were investigated using isolated tryptic peptides of the hormone. Antibodies from virtually all of more than two dozen animals had specificity primarily for the amino-terminal heptadecapeptide (NM) and showed little or no binding with the carboxy-terminal undeca- and dodecapeptides (C). The smallest synthetic peptide of a series initiated at residue 16 which measurably bound antibody comprised residues 5–16 of glucagon. In cellular immune assays, both NM and C elicited delayed cutaneous reactions and inhibited the migration of peritoneal cells from immune animals. However, only intact glucagon and its C fragment stimulated lymphoid cells to synthesize DNA. While glucagon was somewhat more active than C, the addition of NM to C did not enhance its transforming activity. The smallest synthetic carboxy-terminal peptide with discernible transforming activity comprised residues 19–29 of glucagon. In both native and synthetic C peptide preparations, the undecapeptide was generally more active than the dodecapeptide, although cells from different animals gave different response patterns. The difference between the two is the presence of arginine at the amino-terminus of the peptide chain. Thus, the recognition specificity of populations of antigen-reactive cells from different animals displays a variation which is at least superficially analogous to that of populations of antibody molecules. In limited experiments using NM and C peptides as immunogens, neither gave rise to delayed hypersensitivity or to glucagon-binding circulating antibody, following a regimen which invariably provoked these responses when glucagon itself served as the immunogen. These results indicate that glucagon was cleaved by trypsin along functional lines into two parts, one of which housed the major antigenic determinant and the other of which carried the major immunogenic determinant, and they are highly compatible with a two-cell mechanism of immune induction. An apparent dissociation between the capacity to provoke delayed hypersensitivity reactions and to transform antigen-reactive cells in culture was observed.

Studies on Delayed-Type Hypersensitivity to Hen Egg-White Lysozyme

“…We may not conclude that our results on lysozyme coincide with the findings in glucagon (Senyk et al ., 1971). No peptide from tryptic digest of glucagon could function as an immunogen in either antibody formation or delayed -type hypersensitivity, while both peptides , PP-3 and PP-9, from peptic digest of lysozyme were immunogenic .…”

“…It has been reported that the antigenic site on a molecule recognized by immunocytes for production of humoral antibody is distinct from that for induction of delayed-type hypersensitivity with synthetic polypeptides (Schlossman, Herman and Yaron, 1969), flagellin (Ichiki and Parish, 1972) and glucagon (Senyk et al, 1971) as immunogens. The results with flagellin and its acetoacetylated derivatives as immunogens showed no serological cross reactivity 152 YOKOMURO et HOMMA Vol.…”

“…29 between these antigens but a significant degree of cross reactivity was observed with respect to delayed-type hypersensitivity measured by the footpad swelling technique (Ichiki and Parish, 1972). It was demonstrated with the enzymatically digested products of glucagon that the N-terminal area of the molecule was found to be the antigenic determinant for circulating antibody, while the C-terminal part was recognized by antigen-reactive cells (Senyk et al, 1971). It was recently reported that there was cross reactivity between lysozyme and its reduced,, Scarboxymethylated derivative (CM-lysozyme) in delayed-type skin reaction, but not with respect to humoral antibodies (Thompson, Harris and Benjamini, 1972).…”

The Compartmentalization of Antigen-Reactive Lymphocytes in Desensitized Guinea Pigs

“…Studies showing a dissociation between antigen-induced mediator production in one cell population and proliferation in another population deserve further exploration with a common cell population (22,23).…”

Hapten-reactive inducer T cells. I. Definition of two classes of hapten-specific inducer cells.

“…The repeated failure of antibodies directed against soluble antigen to block T cell proliferation has led to two major hypotheses: (a) B cells and their secreted products, antibodies, recognize epitopes that differ from determinants recognized by inducer T cells (17,18); and (b) antigen processing creates new determinants that are not recognized by antibodies because of structural or conformational changes (9,19,20). We show that antibodies specific for epitopes that are not altered during processing 2 Definition of inducer cells as hapten and conjugate specific refers to specificity of activation rather than specificity of binding.…”