Cloning and characterization of a lung-specific cDNA corresponding to the gamma chain of hepatic fibrinogen.

The gene (gamma FBG) encoding the fibrinogen gamma chain (gamma FBG) has been shown to exhibit both tissue-specific and ubiquitous expression. To confirm the identity of the gamma FBG transcripts expressed in extrahepatic tissue, lung tissue was chosen as a model of extrahepatic gamma FBG gene expression. A ferret lung cDNA clone bank was constructed in lambda gt11 and several positive plaques were isolated using cross-species hybridization with the rat gamma FBG cDNA. Sequence data of the longest clone, designated pFLG gamma 3, was compared at the nucleotide and deduced amino acid (aa) levels with sequences of gamma FBG from other species. The results indicated that the identity of the ferret lung-specific gamma FBG cDNA to pig, rat, bovine and human gamma FBG cDNAs ranged from 78-88%; the similarity of the ferret lung-specific gamma FBG deduced aa sequence ranged from 84-88% across species. Cysteine aa involved in intra- and inter-chain disulfide-bonded secondary and tertiary structure are absolutely conserved in ferret gamma FBG. The putative cell-cell adhesion sites for both platelet alpha IIb beta 3 and intercellular adhesion molecule-1 receptor binding to ferret gamma FBG are > 90% similar to the corresponding sites in the human gamma FBG. The results of Northern hybridization indicated that the ferret lung gamma FBG mRNA was equivalent in size to the liver gamma FBG mRNA; Southern hybridization suggested that ferret gamma FBG is a single-copy gene, as is the gamma FBG of other species. Lung-specific gamma FBG expression was localized to epithelial cells of the large and small airways and chondrocytes by in situ RNA:RNA hybridization. The functional significance of gamma FBG expression in lung is not presently known. Since expression of FBG is up-regulated 2-10-fold in the liver during an inflammatory event, it is possible that lung-specific gamma FBG expression occurs predominantly during lung disease or injury.

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.