Abstract
Serpins are responsible for regulating a variety of proteolytic processes through a unique irreversible suicide substrate mechanism. To discover novel genes regulated by transforming growth factor-beta1 (TGF-beta 1), we performed differential display reverse transcriptase-PCR analysis of NRP-152 rat prostatic epithelial cells and cloned a novel rat serpin that is transcriptionally down-regulated by TGF-beta and hence named trespin (TGF-beta-repressible serine proteinase inhibitor (trespin). Trespin is a 397-amino acid member of the ov-serpin clade with a calculated molecular mass of 45.2 kDa and 72% amino acid sequence homology to human bomapin; however, trespin exhibits different tissue expression, cellular localization, and proteinase specificity compared with bomapin. Trespin mRNA is expressed in many tissues, including brain, heart, kidney, liver, lung, prostate, skin, spleen, and stomach. FLAG-trespin expressed in HEK293 cells is localized predominantly in the cytoplasm and is not constitutively secreted. The presence of an arginine at the P1 position of trespin's reactive site loop suggests that trespin inhibits trypsin-like proteinases. Accordingly, in vitro transcribed and translated trespin forms detergent-stable and thermostable complexes with plasmin and elastase but not subtilisin A, trypsin, chymotrypsin, thrombin, or papain. Trespin interacts with plasmin at a near 1:1 stoichiometry, and immunopurified mammal-expressed trespin inhibits plasmin in a dose-dependent manner. These data suggest that trespin is a novel and functional member of the rat ov-serpin family.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY075037
Cloning of Trespin—Differential display RT-PCR was used to identify and isolate genes regulated by TGF-1 in NRP-152 cells. This was done with RNA isolated from NRP-152 cells treated with or without 10 ng/ml TGF-1 for 24 h either alone or in the presence of 5 g/ml insulin, which blocks several effects of TGF-1.2 Following differential display screening with 20 primer sets, we identified the 3Ј-end of a novel gene, which we initially named TGF--regulated gene 13 (TRG-13)
Expression of TRG-13 is decreased by TGF-1 in a manner that is blocked in the presence of insulin (Fig. 1A, doublet is observed due to denaturing electrophoresis of double-stranded DNA)
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY075037. Serpins are identified by their unique conformation [3, 4], namely a conserved secondary structure containing three -sheets (designated A, B, and C), ␣-helices (usually nine), and a reactive site loop (RSL), which confers specificity to proteinase recognition. Clade B members, the ov-serpins, were originally identified by their significant sequence homology to chicken ovalbumin [12]. They are competitive inhibitors of serine or cysteine proteinases and can target more than one proteinase through the use of several P1 residues [13, 14]. The CD loop confers unique characteristics to serpins, such as nuclear localization, and provides a binding motif for ancillary proteins [15,16,17,18].
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