Enzymatic Activity of the Staphylococcus aureus SplB Serine Protease is Induced by Substrates Containing the Sequence Trp-Glu-Leu-Gln

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

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Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design

Protein Engineering Design and Selection ◽

10.1093/protein/gzaa018 ◽

2019 ◽

Vol 32 (12) ◽

pp. 555-564

Author(s):

Magdalena Wójcik ◽

Susana Vázquez Torres ◽

Wim J Quax ◽

Ykelien L Boersma

Keyword(s):

Staphylococcus Aureus ◽

Amino Acid ◽

Enzymatic Activity ◽

Sortase A ◽

Wild Type ◽

Protein Thermostability ◽

Consensus Analysis ◽

Gram Positive Bacteria ◽

Covalently Linked

Abstract Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme’s stability while at the same time enhancing the enzyme’s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

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Staphylococcus aureus Induces Increased Serine Protease Activity in Keratinocytes

Journal of Investigative Dermatology ◽

10.1016/j.jid.2016.10.008 ◽

2017 ◽

Vol 137 (2) ◽

pp. 377-384 ◽

Cited By ~ 46

Author(s):

Michael R. Williams ◽

Teruaki Nakatsuji ◽

James A. Sanford ◽

Alison F. Vrbanac ◽

Richard L. Gallo

Keyword(s):

Staphylococcus Aureus ◽

Serine Protease ◽

Protease Activity ◽

Serine Protease Activity

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Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation

Mediators of Inflammation ◽

10.1155/2012/456462 ◽

2012 ◽

Vol 2012 ◽

pp. 1-13 ◽

Cited By ~ 15

Author(s):

Riyo Morimoto-Kamata ◽

Sei-ichiro Mizoguchi ◽

Takeo Ichisugi ◽

Satoru Yui

Keyword(s):

Breast Cancer ◽

Cell Surface ◽

Enzymatic Activity ◽

Serine Protease ◽

Human Breast Cancer ◽

Cell Aggregation ◽

Catalytic Site ◽

Cathepsin G ◽

Human Breast ◽

Mcf 7

Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG) is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering) CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

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Staphylococcal Exfoliative Toxins Cleave α- and β-Melanocyte-Stimulating Hormones

Infection and Immunity ◽

10.1128/iai.68.4.2366-2368.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 2366-2368 ◽

Cited By ~ 17

Author(s):

James V. Rago ◽

Gregory M. Vath ◽

Timothy J. Tripp ◽

Gregory A. Bohach ◽

Douglas H. Ohlendorf ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Glutamic Acid ◽

Crystal Structures ◽

Serine Protease ◽

Protease Activity ◽

Staphylococcal Scalded Skin Syndrome ◽

Melanocyte Stimulating Hormone ◽

Causative Agents ◽

Serine Protease Family ◽

Stimulating Hormone

ABSTRACT The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains ofStaphylococcus aureus and are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide β-melanocyte-stimulating hormone (β-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving α-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving β-MSH.

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Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing

Journal of Biological Chemistry ◽

10.1074/jbc.m705672200 ◽

2007 ◽

Vol 282 (47) ◽

pp. 34129-34138 ◽

Cited By ~ 31

Author(s):

Nicholas N. Nickerson ◽

Lata Prasad ◽

Latha Jacob ◽

Louis T. Delbaere ◽

Martin J. McGavin

Keyword(s):

Staphylococcus Aureus ◽

Serine Protease

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Nasal Colonization of Serine Protease esp - Positive Staphylococcus epidermidis Affecting Staphylococcus aureus Colonization

Annals of Clinical Microbiology ◽

10.5145/acm.2021.24.3.5 ◽

2021 ◽

Vol 24 (3) ◽

Keyword(s):

Staphylococcus Aureus ◽

Serine Protease ◽

Staphylococcus Epidermidis ◽

Nasal Colonization

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Description of Staphylococcus Serine Protease (ssp) Operon in Staphylococcus aureus and Nonpolar Inactivation of sspA-Encoded Serine Protease

Infection and Immunity ◽

10.1128/iai.69.1.159-169.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 159-169 ◽

Cited By ~ 89

Author(s):

Kelly Rice ◽

Robert Peralta ◽

Darrin Bast ◽

Joyce de Azavedo ◽

Martin J. McGavin

Keyword(s):

Staphylococcus Aureus ◽

Serine Protease ◽

Cysteine Protease ◽

Culture Supernatant ◽

Pleiotropic Effect ◽

Infection Model ◽

Gene Encoding ◽

Growth And Survival ◽

In Vivo Growth

ABSTRACT Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureusRN6390. The ssp gene encoding V8 protease is designated assspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

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