Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor.

Abstract

A Xgtll expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the ,8-migrating plasminogen activator inhibitor (p-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 x i0-phages. Three clones (X1.2, X3, and X9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for A9.2, but not for Xgtll, produced a fusion protein of 180 kDa that was recognized by afflinity-purified antibodies against the bovine aortic endothelial cell fl-PAI and had fl-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human fl-PAL. Based on this alignment, the mature human P-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with ao-antitrypsin and antithrombin III, indicating that the fl-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.The generation of plasmin from plasminogen provides an important source of proteolytic activity in cells, tissues, and biological fluids (1, 2). Precise regulation of plasminogen activator (PA) activity may thus constitute a critical feature of many biological systems (3). Such control may be at the level of the formation and resolution of fibrin itself (4) To facilitate the precise biochemical characterization of P-PAI, and to eventually understand the nature of factors regulating 3-PAI gene expression, we have undertaken the molecular cloning of the gene. Here we describe the isolation of P-PAI cDNA from a human placental expression library and demonstrate that the (3-PAI is a member of the serine protease inhibitor (serpin) superfamily (23, 24).

Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma

“…This is in agreement with the known amino acid sequence of PAl: Val-His-His-ProPro-Ser-Tyr-Val- [4] and that of vitronectin: AspGln-Glu-Ser-Cys-Lys-Gly-Arg- [12].…”

“…In contrast, functionally inactive or so called 'latent' PAl filters as a 50 kDa compound [2,3], in agreement with its suggested primary structure [4]. Recently we presented evidence that the deviating gel filtration behaviour of functionally active PAI in the presence of plasma/serum is due to the existence of a discrete PAl-binding protein in plasma/serum [3].…”

Plasminogen activator inhibitor 1 and 2 are tumor necrosis factor/cachectin-responsive genes.

“…Northern blot analysis indicated that, in a dose-dependent manner, TNF increased PAI-2 mRNA, as well as the 3.4-and 2 .4-bp species (17) of PAI-1 mRNA, while at the same time suppressing t-PA mRNA (Fig . 1) .…”

Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasma plasminogen activator inhibitor-1 activity.

“…A 2.9-kilobase (kb) PAI-1 cDNA probe described previously 24 was used in hybridization analysis and was kindly provided by Tor Ny, University of Umea, Sweden. The Hindlll polymorphism was that first described by Klinger et al 15 DNA was prepared from blood using standard techniques.…”