Factor VII Activating Protease (FSAP): Functional Implications of the “Marburg-1” Polymorphism.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2126-2126
Author(s):  
Fabian Stavenuiter ◽  
Alexander B Meijer ◽  
Erica Sellink ◽  
Koen Mertens
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Proteolytic Processing ◽  
Factor Vii ◽  
Maximal Effect ◽  
Single Chain ◽  
Surface Binding ◽  
Positive Effects ◽  
Charged Surfaces ◽  
Hydrolysis Of

Abstract Abstract 2126 Poster Board II-104 Introduction FSAP is a plasma serine protease first reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and Factor VII (FVII), suggesting a key role in hemostasis and thrombosis. Numerous additional functions have been proposed, including inhibition of smooth muscle cell proliferation and migration. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources due to autocatalytic activity which is stimulated through interaction with negatively charged surfaces. About 5-10% of healthy individuals carry a polymorphism (Marburg-1) at position c221 (G534Q) located in one of the 8 surface binding loops of the serine protease domain. This polymorphism has been proposed to be associated with impaired activation of scuPA in vitro, suggesting a putative defect in fibrinolysis. Epidemiological studies have remained inconclusive with regard to prothrombotic implications of this polymorphism. Residue c221 has been described as highly important in serine proteases. For prothrombin the D221Q mutation has been associated with a severe defect in fibrinogen clotting. Similarly, patients who are hemizygous for a c221 substitution in FIX (A221V) suffer from haemophilia B. In general, in Na+ -dependent serine proteases like FVII, FIX, and thrombin, residue c221 contributes to activity and substrate specificity. Objectives: Our aim was to investigate, using intact recombinant (r) FSAP, the effect of the M1-polymorphism on FSAP biological activity. Results Various stable cell lines (HEK293-, BHK-, LOVO-, and CHO cells) expressing normal rFSAP (wt) and its Marburg-1 (M1) variant were produced. Irrespective to the cell type used, rFSAP was found to be cleaved after expression due to autocatalytic cleavage. However, wtFSAP was found to be more sensitive to proteolytic processing than its M1-variant. Moreover, wtFSAP was found to be completely inactivated whereas the M1-variant could be purified in its two-chain form. To overcome the problem of autocatalytic degradation, for wtFSAP we constructed a FSAP-variant in which the natural activation site (R313-I314) was replaced by a cleavage site for the bacterial protease thermolysin. Thermolysin-activated rFSAP displayed the same affinity for chromogenic peptide substrates (S2288) as pdFSAP (Km 0.38 mM) and retained its capability to activate scuPA (Km 62 nM). Vmax for scuPA activation was increased through interaction with negative charged surfaces like polyphosphate and heparin (2- and 3-fold, respectively), whereas no effect on the hydrolysis of S2288 was found. In contrast, the M1-variant displayed severely reduced affinity for S2288 (6.5-fold) and hardly any scuPA activation. Interestingly, addition of heparin or polyphosphate showed positive effects on the hydrolysis of both substrates by the M1-variant. Compared to wtFSAP, however, both the Km and Vmax were still heavily affected. Surprisingly, wtFSAP proved incapable of cleaving purified FVII, even in the presence of calcium-ions and lipid vesicles of varying composition, including up to 40 mol% negative phospholipids such as phosphatidylserine and cardiolipin (CL). On membranes of 100% CL FVII cleavage did occur, but this resulted in transient activation and rapid degradation. The M1-variant, however, displayed no FVII cleavage under any of the conditions tested. Finally, we found that Na+, in absence of CaCl2, affects the maximal rate of S2288 hydrolysis by rFSAP, with a maximal effect at physiological relevant concentrations. The Na+ concentrations needed to reach maximal catalytic activity of the M1-variant were found 8 - 10 fold above physiologically relevant levels. Conclusions While rFSAP indeed activates scuPA, FVII appears surprisingly resistant to activation by rFSAP. The M1-variant does not activate FVII either, but does display reduced scuPA activation. The M1-polymorphism, being a Gly to Glu substitution at position c221, makes the protease less responsive to Na+. This is compatible with its location in the putative Na+-binding loops. Whether or not the reduced scuPA activation has any physiological impact remains unclear. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment

2010 ◽  
Vol 428 (3) ◽  
pp. 325-346 ◽  
Author(s):  
Toni M. Antalis ◽  
Marguerite S. Buzza ◽  
Kathryn M. Hodge ◽  
John D. Hooper ◽  
Sarah Netzel-Arnett
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Proteolytic Processing ◽  
Coat Proteins ◽  
Vital Functions ◽  
Human Pathology ◽  
Functional Consequences ◽  
Viral Coat ◽  
Precursor Molecules

The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Members of this family contain N- or C-terminal domains that serve to tether the serine protease catalytic domain directly to the plasma membrane. These membrane-anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homoeostasis. Substrates activated by membrane-anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. The present review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease.

scholarly journals Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface

2010 ◽  
Vol 430 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Marit Stirnberg ◽  
Eva Maurer ◽  
Angelika Horstmeyer ◽  
Sonja Kolp ◽  
Stefan Frank ◽  
...  
Keyword(s):  
Cell Surface ◽  
Disulfide Bonds ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Bond Cleavage ◽  
Proteolytic Processing ◽  
Cleavage Sites ◽  
Single Chain

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

LIPOPOLYSACCHARIDE-SENSITIVE SERINE-PROTEASE ZYMOGEN (FACTOR C) OF LIMULUS HEMOCYTES: IDENTIFICATION AND ALIGNMENT OF PROTEOLYTIC FRAGMENTS PRODUCED DURING THE ACTIVATION SHOW THAT IT IS A NOVEL TYPE OF SERINE-PROTEASE

1987 ◽  
Author(s):  
F Tokunaga ◽  
T Miyata ◽  
T Nakamura ◽  
T Morita ◽  
S Iwanaga
Keyword(s):  
Serine Protease ◽  
Heavy Chain ◽  
Light Chain ◽  
Interleukin 2 ◽  
Coagulation Factor ◽  
Clotting Factor ◽  
Single Chain ◽  
Terminal Sequence ◽  
A Chain ◽  
Factor C

Limulus clotting factor, factor C, is a lipopolysaccharide (LPS)-sensitive serine-protease zymogen present in the hemocytes. It is a two-chain glycoprotein (M.W. = 123,000) composed of a heavy chain (M.W. = 80,000) and a light chain (M.W. = 43,000) T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521 .On further studies of this zymogen, a single-chain factor C (M.W. = 123,000) was identified by Western blotting technique. The heavy chain had an NH2-terminal sequence of Ser-Gly-Val-Asp-, which was consistent with the NH2-terminal sequence of the single-chain factor C, indicating that the heavy chain is located in the NH2-terminal part of the zymogen. The light chain had an NH22-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with LPS resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72.amino acids) and B chains derived from the light chain was formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar structuraly to those a family of repeats in human β2 -glycoprotein I, complement factors B, Clr, Cls, H, C4b-binding protein, 02, coagulation factor XIII b subunit, haptoglobin a chain, and interleukin 2 receptor. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence of -ASP-Ala-Cys-Ser-Gly-Asp-SER-Gly-Gly-Pro-.These results indicate that limulus factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine-protease by hydrolysis of a specific Phe-Ile peptide bond. The correlation of limulus factor C and mammalian complement proteins was also suggested.

scholarly journals Carbohydrate-controlled serine protease inhibitor (serpin) production in Bifidobacterium longum subsp. longum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. Duboux ◽  
M. Golliard ◽  
J. A. Muller ◽  
G. Bergonzelli ◽  
C. J. Bolten ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Proteases ◽  
Intestinal Tract ◽  
Defense Mechanism ◽  
Inflammatory Diseases ◽  
Bifidobacterium Longum ◽  
Serine Protease Inhibitor ◽  
Innate Defense

AbstractThe Serine Protease Inhibitor (serpin) protein has been suggested to play a key role in the interaction of bifidobacteria with the host. By inhibiting intestinal serine proteases, it might allow bifidobacteria to reside in specific gut niches. In inflammatory diseases where serine proteases contribute to the innate defense mechanism of the host, serpin may dampen the damaging effects of inflammation. In view of the beneficial roles of this protein, it is important to understand how its production is regulated. Here we demonstrate that Bifidobacterium longum NCC 2705 serpin production is tightly regulated by carbohydrates. Galactose and fructose increase the production of this protein while glucose prevents it, suggesting the involvement of catabolite repression. We identified that di- and oligosaccharides containing galactose (GOS) and fructose (FOS) moieties, including the human milk oligosaccharide Lacto-N-tetraose (LNT), are able to activate serpin production. Moreover, we show that the carbohydrate mediated regulation is conserved within B. longum subsp. longum strains but not in other bifidobacterial taxons harboring the serpin coding gene, highlighting that the serpin regulation circuits are not only species- but also subspecies- specific. Our work demonstrates that environmental conditions can modulate expression of an important effector molecule of B. longum, having potential important implications for probiotic manufacturing and supporting the postulated role of serpin in the ability of bifidobacteria to colonize the intestinal tract.

scholarly journals Pharmacological Characterisation of Pseudocerastes and Eristicophis Viper Venoms Reveal Anticancer (Melanoma) Properties and a Potentially Novel Mode of Fibrinogenolysis

2021 ◽  
Vol 22 (13) ◽  
pp. 6896
Author(s):  
Bianca op den Brouw ◽  
Parviz Ghezellou ◽  
Nicholas R. Casewell ◽  
Syed Abid Ali ◽  
Behzad Fathinia ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Serine Proteases ◽  
Human Melanoma ◽  
Snake Venoms ◽  
Lead Compounds ◽  
Bioactive Properties ◽  
Fibrinogenolytic Activity ◽  
Pharmacological Potential ◽  
Hydrolysis Of ◽  
Viper Venoms

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

ChemInform Abstract: Hydrolysis of 2,4-Dinitrophenyl Phosphate Catalyzed by Single-Chain, Twin-Tailed and Bolaform Surfactants.

ChemInform ◽  
1990 ◽  
Vol 21 (23) ◽  
Author(s):  
C. A. BUNTON ◽  
E. L. DORWIN ◽  
G. SAVELLI ◽  
V. C. SI
Keyword(s):  
Single Chain ◽  
Hydrolysis Of

scholarly journals The Mouse Testis Is the Source of Various Serine Proteases and Serine Proteinase Inhibitors (SERPINs): Serine Proteases and SERPINs Identified in Leydig Cells Are under Gonadotropin Regulation

Endocrinology ◽  
2006 ◽  
Vol 147 (9) ◽  
pp. 4374-4383 ◽  
Author(s):  
Fanny Odet ◽  
Adélie Verot ◽  
Brigitte Le Magueresse-Battistoni
Keyword(s):  
Extracellular Matrix ◽  
Plasminogen Activator ◽  
Serine Proteases ◽  
Leydig Cells ◽  
Proteinase Inhibitors ◽  
Primary Cultures ◽  
Serine Proteinases ◽  
Rt Pcr ◽  
Positive Effects ◽  
Extracellular Matrix Components

The occurrence of various serine proteinases and serine proteinases inhibitors (SERPINs) was investigated by RT-PCR in whole testes of 1-, 3-, and 8-wk-old mice in crude and enriched germ cell fractions, mouse Leydig tumor cells (mLTC-1), and primary cultures of 3- and 8-wk-old enriched fractions of Leydig cells and 3-wk-old Sertoli cells. New members were identified in the testis protease repertoire. Within the Leydig repertoire, a PCR product was found for plasminogen activators urokinase plasminogen activator (uPA) and tissue plasminogen activator (8-wk-old cells), matriptase-2 (mLTC-1), kallikrein-21, SERPINA5, SERPINB2 (primary cultures), and serine peptidase inhibitor Kunitz type 2 (SPINT2). The gonadotropin regulation was explored by semiquantitative RT-PCR, using steroidogenic acute regulatory protein (StAR) as a positive control. Matriptase-2, kallikrein-21, SPINT2, and SERPINA5 were down-regulated, whereas uPA and its receptor were up-regulated by human chorionic gonadotropin (hCG) via cAMP in the mLTC-1 cells. Positive effects were observed transiently after 1–8 h of hCG exposure, and negative effects, first evidenced after 6 h, lasted 48 h. The hCG-induced effects were confirmed in primary cultures. In addition, SERPINB2 was augmented by hCG in primary cultures. Addition of either trypsin or protease inhibitors did not alter the hCG-induced surge of StAR. Because hCG regulated proteases and SERPINs (whereas testosterone did not), it could alter the proteolytic balance of Leydig cells and consequently the metabolism of extracellular matrix components. Therefore, even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely, our data together with the literature suggest that extracellular matrix proteins alter Leydig cell steroidogenesis.

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