Molecular cloning, expression, and characterization of bovine tissue factor pathway inhibitor-2.
Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.
Du
Xin
X
Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Deng
Fang-Ming
FM
Chand
Hitendra Singh
HS
Kisiel
Walter
W
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004305:Dose-Response Relationship, Drug; D015942:Factor VIIa / Q000037:antagonists & inhibitors; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000187:drug effects; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014357:Trypsin / Q000187:drug effects / Q000378:metabolism
D006023:Glycoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C021240:tissue-factor-pathway inhibitor 2; D015942:Factor VIIa; D014357:Trypsin; D005341:Fibrinolysin