Cloning of the complete coding sequence of rat fibrinogen B beta chain cDNA: interspecies conservation of fibrin beta 15-42 primary structure.
After thrombin cleavage, the newly exposed NH2-termini of the beta chains play a role in both fibrin polymerization and fibrin interactions with cells in the process of wound healing. These physiological responses have been shown to be mediated, at least in part, by beta 15-42. To compare the sequence of the beta chain of fibrin across species, the complete coding sequence of the rat fibrinogen B beta chain cDNA was cloned (designated pRB beta 3) and characterized. The sequence newly determined from pRB beta 3 encompassed nucleotides 121-589, encoding residues 9-165 of the mature polypeptide. Significant homology of pRB beta 3 cDNA and deduced amino acid sequences was found when compared with other species' B beta chains. The rat B beta-Arg14-Gly15 thrombin cleavage site is conserved; however, the fibrinopeptide B sequences are only 50% similar when rat is compared with human. In contrast, the beta 15-42 region is 100% similar when allowing for conservative amino acid substitutions. Monoclonal antibodies (MAbs) specific for human fibrinogen B beta 1-21 (1-8C6) and human fibrin beta 15-21 (59D8 and T2G1) failed to cross-react with rat fibrinogen or fibrin by ELISA, respectively, even though thrombin conversion of rat fibrinogen to fibrin was confirmed. MAb 1-8C6 reacted with reduced and denatured human fibrinogen B beta chain by Western blotting, whereas, MAb T2G1 did not blot with reduced and denatured human fibrin beta chain. A comparative analysis of the binding affinity of the human B beta fibrin(ogen) specific MAbs with B beta fibrin(ogen) from several species suggested that amino acid residues preceding and including Arg14-Gly15 are important in the epitope of the B beta 1-21 specific MAb 1-8C6, and that residues Gly15, Leu19 and/or Lys21 play an important role in the epitope shared by the beta 15-21-specific MAbs T2G1 and 59D8.
Department of Medicine/Hematology Unit, University of Rochester School of Medicine and Dentistry, NY 14642.
M A
MA
Courtney
L A
LA
Bunce
L A
LA
Neroni
P J
PJ
Simpson-Haidaris
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry; D005340:Fibrinogen / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats / Q000235:genetics*; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D013045:Species Specificity
D018076:DNA, Complementary; D005338:Fibrin Fibrinogen Degradation Products; D010446:Peptide Fragments; C033786:fibrinogen Bbeta (15-42); D005340:Fibrinogen