Chemical cross-linking of activated coagulation factor VII with soluble tissue factor: calcium ions are not essential for full amidolytic activity of the factor VIIa-tissue factor complex after complex formation.
In the previous study involving a yeast expression system, a high molecular mass extracellular domain of human tissue factor (denoted as sTF alpha) with a high content of mannose residues was produced in abundance and 37 kDa sTF beta was obtained in a low yield [Shigematsu et al. (1992) J. Biol. Chem. 267, 21329-21337]. To obtain sTF beta in a high yield, we constructed four kinds of mutant sTF with partial or total replacement of the N-potential glycosylation Asn residues with Ala, and expressed them in yeast. We found that the yield of the beta form of the Asn137-to-Ala mutant (designated as sTF beta NNA) was threefold higher (3 mg/liter) than that of the wild type, suggesting that the replacement of one of the three potential N-glycosylation Asn residues with Ala could be a good way to minimize the addition of mannose repeats. Since it has been reported that calcium ions are required for the effective hydrolysis of peptidyl substrates by the factor VIIa-sTF complex, it is believed to be essential for the expression of full protease activity. Here, we report the enzymatic characterization of a factor VIIa-sTF beta NNA complex cross-linked with a homobifunctional reagent, bis(sulfosuccinimidyl) suberate. The factor VIIa-sTF beta NNA complex cross-linked in the presence of 5 mM calcium ions or 50 mM EDTA was purified. The cross-linked complex did not show factor X activation in the presence of phospholipids. However, it showed essentially the same activity toward peptidyl substrates as before cross-linking, even in the presence of EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)
Miyata
T
T
National Cardiovascular Center Research Institute, Osaka.
Funatsu
A
A
Kato
H
H
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D001777:Blood Coagulation; D002118:Calcium / Q000737:chemistry* / Q000494:pharmacology / Q000502:physiology*; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology*; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015951:Factor Xa / Q000096:biosynthesis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012995:Solubility; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D000577:Amides; D003432:Cross-Linking Reagents; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa; D002118:Calcium