Molecular Cloning of Bomapin (Protease Inhibitor 10), a Novel Human Serpin That Is Expressed Specifically in the Bone Marrow

Abstract

Serine proteinase inhibitors or serpins are a superfamily of homologous proteins that are for the most part involved in the regulation of proteolytic processes in a variety of biological systems. Utilizing a polymerase chain reaction-based strategy we have cloned a novel member of the ovalbumin family of serpins from a human bone marrow cDNA library. The new gene encodes a 397-amino acid protein, designated bomapin, with a calculated molecular mass of 45 kDa and 48% amino acid identity with plasminogen activator inhibitor-2, human leukocyte elastase inhibitor, and cytoplasmic antiproteinase. A single 2.3-kilobase bomapin transcript is highly expressed in human bone marrow cells but was undetectable in all other analyzed human tissues. In vitro transcription and translation of the bomapin cDNA revealed the synthesis of an appropriately sized protein that was able to form SDS-stable complexes with thrombin and trypsin. The restricted expression of bomapin to the bone marrow raises the possibility that this serpin may play a role in the regulation of protease activities during hematopoiesis.Serine proteinase inhibitors or serpins are a ubiquitous superfamily of homologous proteins that resemble ␣ 1 -proteinase inhibitor (␣ 1 -PI) 1 in overall structure (1). In general, serpins contain a highly exposed reactive site loop near the C terminus of the molecule that interacts as a pseudosubstrate for the target protease. The inhibitory specificity of the serpin is mainly defined by the P 1 -P 1 Ј amino acid residues in the reactive site loop. Interaction of these amino acids with the active site of the target protease triggers a dramatic conformational change in the serpin that results in a stoichiometric 1:1 inhibitory complex with the protease, which is typically stable to treatment with denaturants such as SDS (2). In addition to the serpins that regulate protease activity, several members of this superfamily lack a protease inhibitory capability, e.g. angiotensinogen and ovalbumin (1, 3). Ovalbumin represents the parent prototype of a unique family of proteins within the serpin superfamily, which have several structural features in common, including the absence of a typical cleavable signal sequence (4). Previously identified members of this ovalbumin family of serpin proteins (ov-serpins) include plasminogen activator inhibitor-2 (PAI-2) (5), human leukocyte elastase inhibitor (6), and squamous cell carcinoma antigen (7). More recently, a tumor suppressor called maspin (8), which is presumably not a protease inhibitor (9, 10), and cytoplasmic antiproteinase or CAP (11), also known as placental thrombin inhibitor (12) or as protease inhibitor 6 (13), have been identified as ov-serpins.Although cytoplasmic proteases play a key role in a variety of cellular functions (14, 15), little is known about the physiological targets of the ov-serpins that are expressed predominantly or exclusively as intracellular proteins (i.e. PAI-2, human leukocyte elastase inhibitor, and CAP). Kumar and Baglioni (16) have shown that overe...

A New Family of 10 Murine Ovalbumin Serpins Includes Two Homologs of Proteinase Inhibitor 8 and Two Homologs of the Granzyme B Inhibitor (Proteinase Inhibitor 9)

“…Although otherwise identical in structure, the PI-6 gene lacks an intron present in PAI-2, SCCA-1, and SCCA-2 (8,21). By amino acid sequence, PI-8 and PI-9 are more like PI-6 (13), whereas PI-10 resembles PAI-2 (15).…”

Identification of a Nuclear Targeting Domain in the Insertion between Helices C and D in Protease Inhibitor-10

“…During studies investigating the presence of protease inhibitors in hematopoiesis, our group utilized a polymerase chain reaction (PCR)-based homology cloning strategy to identify a novel ov-serpin, which exhibited a high amino acid homology (48%) with PAI-2, protease inhibitor-6, and human leukocyte elastase inhibitor (13). The isolated cDNA contains a single large open reading frame that encodes a 397-amino acid protein (13).…”

Protease Inhibitor 10 Inhibits Tumor Necrosis Factor α-induced Cell Death

“…Plasmid Constructions-The coding region for PI10 (1) and ␤-galactosidase (␤-gal) (pIND/lacZ; Invitrogen, Carlsbad, NM) were, respectively, subcloned into a NheI/XhoI-or a NheI/NotI-digested pCI-neo expression vector (Promega, Madison, WI). pEGFP/PI10 and pEGFP/ PAI2 expression constructs were prepared as described previously (3).…”

“…Protease inhibitor 10 (PI10) 1 (1) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that exhibits an expression pattern restricted to cells within the monocytic lineage (2). The absence of a signal leader sequence in PI10's coding sequence (1,2) coupled with a nuclear localization signal in its C-D interhelical loop (3) results in this inhibitor being present both as a cytoplasmic and a nuclear molecule.…”

“…The absence of a signal leader sequence in PI10's coding sequence (1,2) coupled with a nuclear localization signal in its C-D interhelical loop (3) results in this inhibitor being present both as a cytoplasmic and a nuclear molecule. The unique tissue and subcellular distribution for this serpin raises the possibility that PI10 may play a key role in normal hematopoietic development, a process that requires the regulation of cell viability, growth, and differentiation.…”