SERPINB12 Is a Novel Member of the Human ov-serpin Family That Is Widely Expressed and Inhibits Trypsin-like Serine Proteinases

Abstract

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 ؎ 1.6 ؋ 10 5 and 1.6 ؎ 0.2 ؋ 10 4 M ؊1 s ؊1 , respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.The serpin 1 superfamily contains over 500 members and has representatives in the genomes of most metazoa, plants, and certain viruses (reviewed in Ref. 1). Family members are easily identified by amino acid sequence alignments due to their high degree of structural conservation. The serpin tertiary structure consists of three ␤-sheets, approximately nine ␣-helices, and several loops that are arranged into a metastable conformation. Serpins employ a unique suicide-substrate-like inhibitory mechanism to neutralize their target proteinases. The mobile reactive site loop (RSL), which is perched on the surface of the molecule, serves as the pseudo-substrate and binds to the active site of the proteinase. Upon RSL cleavage, the serpin undergoes a major conformational rearrangement that traps the proteinase in a covalent acyl-enzyme intermediate (2). Serpins utilize this inhibitory mechanism to regulate proteinase cascades involved in blood clotting, fibrinolysis, complement activation, cell motility, inflammation, and cell death (1, 3, 4). In 1993, Remold-O'Donnell (5) reported on a subset of serpins with a high degree of sequence similarity to chicken ovalbumin. Unlike the canonical serpin ␣1-antitrypsin (SER-PINA1), members of the ov-serpin subfamily lack both classic N-terminal signal peptides and long N-and C-terminal extensions. The ov-serpins also contain a variable length loop between helices C and D that may confer functional motifs involved in, for example, nuclear localization (6) or transglutamination (7). Also, most of the ov-serpins appear to reside intracellularly with...

SERPINB11 Is a New Noninhibitory Intracellular Serpin

“…Determination of Protein Concentrations-Trypsin and plasmin were active site-titrated using 4-methylumbelliferyl-para-guanidinobenzoate (Sigma) as described (25). The concentrations of recombinant serpins were determined by using the Bio-Rad protein assay kit II (Bio-Rad).…”

“…Mouse Orthologue-To identify novel clade B genomic sequences, we used the BLAST2 algorithm and exon 8 of SER-PINB4 to query the human genomic DNA sequence deposited in GenBank TM (25). This exon was chosen, since it contained the highly conserved hinge region of the RSL (28) and the PROSITE serpin signature motif.…”

Identification and characterization of a new member of serpin family- HongrES1 in rat epididymis

“…In mammals, serpin s are expressed in many tissues and they regulate the activities of a diverse array of serine and cysteine proteases, controlling complement activation, blood coagulation, fibrinolysis, inflammation, tumor cell metastasis, apoptosis, neurite extension, and many other physiological processes [5]. And it was also reported that several serpin s play important roles in male reproduction [6][7][8].…”

Intracellular and extracellular serpins modulate lung disease

“…83,84 SERPINB12 inhibits trypsin and is expressed in the lung. 85 Together, the clade B serpin genes encode proteins with diverse antipeptidase activity, with critical intracellular roles in protecting cells from damage, and maintaining normal function in the lung.…”