H. Furthmayr

Evidence for two antigenic determinants in the C-terminal region of rat skin collagen

The ability of the antigenic determinants of collagen to react with antibodies does not depend on the intact triplehelical structure. This property qualifies collagen for a comprehensive immunochemical investigation. Recent success in the chemical characterization of peptides obtained by cleavage with cyanogen bromide [l ,2] or with collagenase [3] has favoured such studies. Thus, Pontz et al. [4] were able to localize two different antigenic determinants on the C-terminal and on a middle region, respectively, of the calf skin collagen molecule. Michaeli et al. [S] assigned an antigenic determinant to the N-terminal region of guinea-pig collagen. In these studies, rabbit antibodies have been employed and it may be predicted that both calf skin and guinea-pig collagen should differ in their sequence from rabbit collagen at least at the antibody-recognition sites. A consistent application of immunological methods should highly facilitate the localization and elucidation of structural variations among different collagens and might also lead to a better understanding of the phenomenon of immunological discrimination. For these reasons some of the antigenic determinants of rat skin collagen were characterized by the same methods as already applied to calf collagen [4].

Different antigenic determinants in the polypeptide chains of human collagen.

Antibodies should provide a valuable tool to compare human collagen from different tissues as well as collagen obtained from normal or pathologic sources. To draw reasonable conclusions from such studies a detailed knowledge is required on the sites which react with the antibodies. The antigenic properties of soluble human collagen have already been the subject of several reports [1 -3 ] . Expect for the investigation of Lindsley et al. [3] there was as yet no attempt to characterize the immunologic activity at the molecular level. Recently, distinct groups of antigenic determinants could be localized in the polypeptide chains of various collagens [4 -7 ] . Such attempts became feasible by studying separated polypeptide chains of collagen or even smaller peptides as obtained after cyanogen bromide cleavage. This approach was now applied to collagen from human dura mater. The present study demonstrated an immune response in rabbits to the a l as well as the o~2-chain with an appreciable heterogeneity of the antibody specificities involved. The major antigenic site is localized on the C-terminal cyanogen bromide peptide, al-CB6. Additional antigenic determinants were found in N-terminal or middle regions of the molecule which, however, are recognized either less frequently or only with a low antibody response.

Increased lysine hydroxylation in rat bone and tendon collagen and localization of the additional residues

Abstract The α1 and α2 chain obtained from guanidine extracts of rat bone collagen contain 10 and 6 additional hydroxylysine residues, in comparison to the respective α chains of rat skin collagen. An increase was also demonstrated for insoluble bone collagen. At least for the α1 chains, no evidence was obtained for a corresponding increase in carbohydrate content. Hydroxylysine values intermediate between those of skin and bone are found for the α2 chain of rat tail tendon collagen, yet the α1 chain is in this respect indistinguishable from that of skin. The relationship of the α chains of rat bone to type I collagen was demonstrated by CNBr cleavage and characterization of most of the fragments. This approach also allowed the localization of the additional hydroxylysine residues in three nonhelical cross-linking regions, as well as in various sites of the helical sequence. The findings do not support the idea that glycine in a position adjacent to the carboxyl group of lysine is an absolute requirement for hydroxylation.

Structural requirements of antigenic determinants in the aminoterminal region of the rat collagen α2 chain

Abstract Three overlapping antigenic determinants were defined by the use of several rabbit antisera to soluble rat collagen on the CNBr peptide α2-CB1 containing 14 amino acid residues. In each antigenic determinant the sequence 5 Lys-Gly was essential as well as, with one exception, the participation of a closely located tyrosyl residue. The minimal size involved four to six amino acid residues.

Chicken antibodies to soluble rat collagen—II: Specificity of the reactions with individual polypeptide chains and cyanogen bromide peptides of collagen

Abstract Chicken antibodies to native rat skin collagen reacted with the random-coiled α1- and α2-polypeptide chains in gel precipitation and passive hemagglutination. The chains were not equivalent in their serologic specificity. They could not be distinguished from α-chains derived from pronase-treated collagen which lacks short, terminal sequences. The antibodies reacted with all of the large cyanogen bromide peptides obtained from the α-chains. Evidence is provided that each of these peptides carries unique antigenic determinants not found at other sites of the molecule. Complete serologic identity or strong cross-reactions were observed between α-chains obtained from different rat tissues or from other species. The results suggested that these antigenic determinants are conformation independent and are located in amino acid sequences of the helical part of the collagen molecule, known to be of high evolutionary stability. The more variable, non-helical sequences in the terminal positions of the chains were not involved. These findings are discussed in relation to the quite different specificity reported for rabbit antibodies to rat collagen.