Molecular cloning and biochemical characterization of rabbit factor XI.
Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160 kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80 kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.
Sinha
Dipali
D
The Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, U.S.A. dipali@astro.temple.edu
Marcinkiewicz
Mariola
M
Gailani
David
D
Walsh
Peter N
PN
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005172:Factor XI / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011288:Prekallikrein / Q000737:chemistry; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D018076:DNA, Complementary; D005172:Factor XI; D011288:Prekallikrein; D003545:Cysteine