Processing and expression of rat and human clotting factor-X-encoding cDNAs.

The cDNA encoding clotting factor X, which participates in the middle stage of the blood coagulation cascade was cloned from a rat liver cDNA library. Sequencing of the rat factor-X-encoding cDNA revealed that this vitamin-K-dependent protein has a dibasic Arg-Arg sequence at the propeptide cleavage site, as occurs in other vitamin-K-dependent proteins. Although the human and rat deduced amino acid sequences are remarkably similar (76% identical), they do significantly differ in that human factor-X contains a unique Thr-Arg sequence at the propeptide cleavage site [Fung et al., Proc. Natl. Acad. Sci. USA 82 (1985) 3591-3595], where a dibasic sequence would normally be expected. This specific site is the recognition motif for the endoprotease, furin, which is located in the Golgi apparatus. Both rat and human cDNAs expressed in Cos-1 cells resulted in secretion of a mixture of single- and two-chain forms of factor X. The two-chain forms were devoid of the propeptide and were produced at similar rates by the transfected cells. The efficient processing of human factor X, when compared to rat factor X, may indicate that an additional protease(s), which recognizes the Thr-Arg motif, may be involved in proteolytic processing of the human enzyme.

Characterization of recombinant human coagulation factor XFriuli.

Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational gamma-carboxy-glutamic acid (Gla) and beta-hydroxy aspartic acid (beta-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel beta-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

1H NMR assignment and secondary structure of the Ca2(+)-free form of the amino-terminal epidermal growth factor like domain in coagulation factor X.

Blood coagulation factor X is composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). The N-terminal EGF like domain in factor X (fX-EGFN), residues 45-86 of the intact protein, contains a beta-hydroxylated aspartic acid and has one Ca2(+)-binding site. Using 2D NMR techniques, we have made a full assignment of the 500-MHz 1H NMR spectrum of Ca2(+)-free fX-EGFN. On the basis of this assignment and complementary NOESY experiments, we have also determined the secondary structure of Ca2(+)-free fX-EGFN in water solution. Residues 45-49 are comparatively mobile, whereas residues 50-56 are constrained by two disulfide bonds to one side of an antiparallel beta-sheet involving residues 59-64 and 67-72. Another antiparallel beta-sheet involves residues 76-77 and 83-84. A small, parallel beta-sheet connects residues 80-81 and 55-56 and thereby orients the two antiparallel beta-sheets relative to each other. Four beta-turns are identified, involving residues 50-53, 56-59, 64-67, and 73-76. Residues 78-82 adopt an extended bend structure. On the basis of secondary structure and the location of the three disulfide bonds, we find that Asp 46, Asp 48, and Hya 63 are sufficiently close to each other to form a Ca2(+)-binding site. However, the amino terminus of the Ca2(+)-free form of fX-EGFN is not part of a triple-stranded beta-sheet as in other EGF like peptides. Differences and similarities between fX-EFGN and murine EGF with respect to secondary structure and conformational shifts are discussed.