Complementary DNA Cloning and Kinetic Characterization of a Novel Intracellular Serine Proteinase Inhibitor: Mechanism of Action with Trypsin and Factor Xa as Model Proteinases

Morgenstern, Kurt; Sprecher, Cindy A.; Holth, Laurel T.; Foster, Donald C.; Grant, Francis James; Ching, Andrew; Kisiel, Walter

doi:10.1021/bi00177a037

Cited by 27 publications

(45 citation statements)

References 52 publications

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“…We attempted to quantify the effectiveness of SERP-1 at inhibiting these six enzymes by investigating their reactions with SERP-1 using the techniques of slow binding inhibition kinetics (36,42). Each proteinase was incubated with various concentrations of SERP-1 in the presence of a fixed concentration of an appropriate chromogenic proteinase substrate.…”

Section: Resultsmentioning

confidence: 99%

Inhibitory Specificity of the Anti-inflammatory Myxoma Virus Serpin, SERP-1

Nash¹,

Whitty²,

Handwerker³

et al. 1998

Journal of Biological Chemistry

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SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissuetype plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg 319 -Asn 320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr 315 -Leu 316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from k inh ‫؍‬ 3 ؋ 10 5 M ؊1 s ؊1 with thrombin to ϳ600 M ؊1 s ؊1with C1s, while steady-state inhibition constants ranged from K I ‫؍‬ 10 pM with thrombin to ϳ100 nM with C1s. Stoichiometries of inhibition varied between SI ‫؍‬ 1.4 ؎ 0.1 for uPA to SI ‫؍‬ 13 ؎ 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with K I (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by k inh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.

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Section: Resultsmentioning

confidence: 99%

Inhibitory Specificity of the Anti-inflammatory Myxoma Virus Serpin, SERP-1

Nash¹,

Whitty²,

Handwerker³

et al. 1998

Journal of Biological Chemistry

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“…In contrast, utilizing CAP-specific primers, appropriately sized PCR products (1175 base pairs) were readily detected after amplification from all three cDNA templates, including the brain cDNA. CAP expression in the brain has been observed to be either undetectable (11) or barely detectable (12) by Northern blotting. Although we cannot rule out the possi- Pairwise alignments between ␣ 1 -PI, bomapin, PAI-2, human elastase inhibitor (EI), and CAP were generated using the GAP routine of the Genetics Computer Group (Madison, WI) software package (gap weight, 3.0; length weight, 0.1) and adjusted manually to reduce the number of gaps in the final alignment.…”

Section: Cloning Of Bone Marrow Serpin 26755mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Riewald

Schleef

1995

Journal of Biological Chemistry

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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...

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“…Human members of the ov-serpin family include plasminogen activator inhibitor-2 (PAI-2) (5), an elastase inhibitor isolated from monocyte-like cells (6), squamous cell carcinoma antigen (7), cytoplasmic antiproteinase (i.e. protease inhibitor-6) (8,9), and a tumor suppressor called maspin (10). Two serpins related to protease inhibitor-6 have been cloned from a placental gtII library (i.e.…”

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confidence: 99%