ISOLATION AND PARTIAL STRUCTURAL CHARACTERIZATION OF AN EQUINE FIBRINOGEN CNBR FRAGMENT THAT EXHIBITS IMMUNOLOGICAL CROSS-REACTIVITY WITH AN A-ALPHA-CHAIN CROSS-LINKING REGION OF HUMAN FIBRINOGEN

Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine Aα chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, Aα 241–476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqAαCNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues Aα 241–517 from the COOH-terminal two-thirds of the human Aα chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at Aα 476 in the human Aα chain was replaced by a valine residue, thus accounting, in part, for the larger EqAαCNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to Aα 428–464 in the human Aα chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine Aα chains, indicate that the region corresponding to (human) A′ 240–517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human Aα chain. © 1990, American Chemical Society. All rights reserved.