Characterization of a cDNA coding for human factor X.

Abstract

A .lambda.gt11 c[complementary]DNA library containing DNA inserts prepared from human liver mRNA was screened with an antibody to human factor X, a plasma protein participating in the middle phase of the blood coagulation cascade. Ten positive clones were isolated from 2 .times. 106 phage and plaque purified. The cDNA in the phage containing the largest insert was sequenced and shown to code for human factor X. This cDNA insert contained 1137 base pairs coding for a portion of the L chain of the molecule, a connecting region, the H chain, a stop codon, a short 3'' noncoding region and a poly(A) tail. The sequence of A-T-T-A-A-A, which functions as a potential recognition site for polyadenylylation or processing, was present in the 3'' end of the coding sequence and preceded the stop codon of TGA by 1 base pair and the poly(A) tail by 14 base pairs. The amino acid sequence deduced from the cDNA indicated that factor X is synthesized as a single-chain polypeptide containing the L and H chains connected by an Arg-Lys-Arg tripeptide. The single-chain molecule is then converted to the L and H chains by cleavage of 2 (or more) internal peptide bonds. In plasma, these 2 chains are linked together by a disulfide bond. The DNA sequence coding for the active site of human factor X showed a high degree of identity with prothrombin and factor IX, 2 other vitamin K-dependent Ser proteases that participate in blood coagulation. These data along with the protein sequence data previously published for the L chain of human factor X establish the complete amino acid sequence for the mature protein present in plasma.