Role of each Asn-linked glycan in the anticoagulant activity of human protein C inhibitor.
The N-glycosylation site mutants of human protein C inhibitor (PCI; N230S, N243Q, N319Q, N230S/N243Q, and N230S/N319Q) were prepared by amino acid replacement of the asparagine residue with a serine or glutamine residue using site-directed mutagenesis and expressed in the baculovirus/insect cell expression system. To examine the importance of each Asn-linked glycan in the activity of PCI, we compared wtPCI with the mutants of N-glycosylation site(s) in terms of the procoagulant protease-inhibitory and anticoagulant activities. The inhibitory activities of N230S, N319Q, and N230S/N319Q toward human thrombin and plasma kallikrein were significantly increased compared with wtPCI, but those of N243Q and N230S/N243Q were reduced. The inhibitory activity of N230S toward human plasma coagulation was significantly increased compared with wtPCI, and that of N230S/N319Q was also significantly increased compared with N319Q. Furthermore, the procoagulant protease-inhibitory and anticoagulant activities of N230S/N319Q (glycosylated on Asn243 only) compared favorably with those of N230S, and both of the mutants possessed highest activities in the purified mutants. These results suggest that the Asn243-linked glycan in PCI molecule possesses critical roles for its anticoagulant activity in the circulation, and the Asn230-linked glycan down-regulates the activity of PCI.
Fujita
Mitsugu
M
Development and Research Laboratories, JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan. fujita@jcrpharm.co.jp
Izutani
Wakako
W
Takahashi
Kenichi
K
Nishizawa
Koji
K
Shirono
Hiroyuki
H
Koga
Junichi
J
D019943:Amino Acid Substitution / Q000502:physiology; D001216:Asparagine / Q000502:physiology; D001777:Blood Coagulation / Q000187:drug effects*; D006031:Glycosylation; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000502:physiology*; D018046:Protein C Inhibitor / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D013917:Thrombin / Q000037:antagonists & inhibitors
D011134:Polysaccharides; D018046:Protein C Inhibitor; D015842:Serine Proteinase Inhibitors; D001216:Asparagine; D007610:Kallikreins; D013917:Thrombin