Login Register Cart 0
Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Protealysin is not Secreted Constitutively

Author(s): Ksenia N. Chukhontseva, Vadim V. Salnikov, Oleg S. Morenkov, Sergey V. Kostrov and Ilya V. Demidyuk*

Volume 26, Issue 3, 2019

Page: [221 - 226] Pages: 6

DOI: 10.2174/0929866526666181212114907

Price: $65

Abstract

Background: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published.

Objective: Here, the protealysin cellular localization was studied for the first time using immunochemical methods.

Methods and Results: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm.

Conclusion: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis.

Keywords: Metalloprotease, protealysin, secretion, Serratia proteamaculans, immunoelectron microscopy, Western blotting.

« Previous Next »
Graphical Abstract

[1]
Demidyuk, I.V.; Gasanov, E.V.; Safina, D.R.; Kostrov, S.V. Structural organization of precursors of thermolysin-like proteinases. Protein J., 2008, 27, 343-354.
[2]
Demidyuk, I.V.; Gromova, T.Y.; Kostrov, S.V. Protealysin. In: Handbook of Proteolytic Enzymes, 3rd ed.; Rawlings, N.D.; Salvesen, G., Eds. Academic Press: Oxford. 2013, Vol. 1, pp 507-602.
[3]
Demidyuk, I.V.; Kalashnikov, A.E.; Gromova, T.Y.; Gasanov, E.V.; Safina, D.R.; Zabolotskaya, M.V.; Rudenskaya, G.N.; Kostrov, S.V. Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor. Protein Expr. Purif., 2006, 47, 551-561.
[4]
Bozhokina, E.S.; Tsaplina, O.A.; Efremova, T.N.; Kever, L.V.; Demidyuk, I.V.; Kostrov, S.V.; Adam, T.; Komissarchik, Y.Y.; Khaitlina, S.Y. Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin. Cell Biol. Int., 2011, 35, 111-118.
[5]
Cabral, C.M.; Cherqui, A.; Pereira, A.; Simoes, N. Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29. Appl. Environ. Microbiol., 2004, 70, 3831-3838.
[6]
Held, K.G.; LaRock, C.N.; D’Argenio, D.A.; Berg, C.A.; Collins, C.M. A metalloprotease secreted by the insect pathogen Photorhabdus luminescens induces melanization. Appl. Environ. Microbiol., 2007, 73, 7622-7628.
[7]
Kothary, M.H.; McCardell, B.A.; Frazar, C.D.; Deer, D.; Tall, B.D. Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii. Appl. Environ. Microbiol., 2007, 73, 4142-4151.
[8]
Feng, T.; Nyffenegger, C.; Hojrup, P.; Vidal-Melgosa, S.; Yan, K.P.; Fangel, J.U.; Meyer, A.S.; Kirpekar, F.; Willats, W.G.; Mikkelsen, J.D. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1. Appl. Microbiol. Biotechnol., 2014, 98, 10077-10089.
[9]
Kyostio, S.R.; Cramer, C.L.; Lacy, G.H. Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene. J. Bacteriol., 1991, 173, 6537-6546.
[10]
Kwon, Y.T.; Lee, H.H.; Rho, H.M. Cloning, sequencing, and expression of a minor protease-encoding gene from Serratia marcescens ATCC21074. Gene, 1993, 125, 75-80.
[11]
Matveyev, V.V.; Usmanova, A.M.; Morozova, A.V.; Collins, J.H.; Khaitlina, S.Y. Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain. Biochim. Biophys. Acta, 1996, 1296, 55-62.
[12]
Morozova, A.V.; Skovorodkin, I.N.; Khaitlina, S.Y.; Malinin, A.Y. Bacterial protease ECP32 specifically hydrolyzing actin and its effect on cytoskeleton in vivo. Biochemistry (Mosc.), 2001, 66, 83-90.
[13]
Zamaliutdinova, N.M.; Minnullina, L.F.; Sharipova, M.R.; Mardanova, A.M. New metalloendopeptidase of Morganella morganii ZM. Bioorg. Khim., 2014, 40, 682-687.
[14]
Lennox, E.S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology, 1955, 1, 190-206.
[15]
Gromova, T.Y.; Demidyuk, I.V.; Kozlovskiy, V.I.; Kuranova, I.P.; Kostrov, S.V. Processing of protealysin precursor. Biochimie, 2009, 91, 639-645.
[16]
Stults, N.L.; Asta, L.M.; Lee, Y.C. Immobilization of proteins on oxidized crosslinked Sepharose preparations by reductive amination. Anal. Biochem., 1989, 180, 114-119.
[17]
Schagger, H. Tricine-SDS-PAGE. Nat. Protoc., 2006, 1, 16-22.
[18]
Vicré, M.; Jauneau, A.; Knox, J.P.; Driouich, A. Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues. Protoplasma, 1998, 203, 26-34.
[19]
Gorshkov, V.; Islamov, B.; Mikshina, P.; Petrova, O.; Burygin, G.; Sigida, E.; Shashkov, A.; Daminova, A.; Ageeva, M.; Idiyatullin, B.; Salnikov, V.; Zuev, Y.; Gorshkova, T.; Gogolev, Y. Pectobacterium atrosepticum exopolysaccharides: Identification, molecular structure, formation under stress and in planta conditions. Glycobiology, 2017, 27, 1016-1026.
[20]
Mokshina, N.E.; Ibragimova, N.N.; Salnikov, V.V.; Amenitskii, S.I.; Gorshkova, T.A. Galactosidase of plant fibers with gelatinous cell wall: Identification and localization. Russ. J. Plant Physiol., 2012, 59, 246-254.
[21]
Salnikov, V.V.; Grimson, M.J.; Seagull, R.W.; Haigler, C.H. Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers. Protoplasma, 2003, 221, 175-184.
[22]
Oppermann, M. Anion exchange chromatography for purification of monoclonal IgG antibodies.In: Monoclonal Antibodies; Peters, J.H.; Baumgarten, H., Eds.; Springer: Berlin, Heidelberg, 1992, pp. 271-275.
[23]
Demidyuk, I.V.; Gromova, T.Y.; Kostrov, S.V. The propeptide is required for in vivo formation of active protealysin. Protein Pept. Lett., 2015, 22, 509-513.
[24]
Demidyuk, I.V.; Gromova, T.Y.; Polyakov, K.M.; Melik-Adamyan, W.R.; Kuranova, I.P.; Kostrov, S.V. Crystal structure of the protealysin precursor: Insights into propeptide function. J. Biol. Chem., 2010, 285, 2003-2013.
[25]
Tsaplina, O.A.; Efremova, T.N.; Kever, L.V.; Komissarchik, Y.Y.; Demidyuk, I.V.; Kostrov, S.V.; Khaitlina, S.Y. Probing for actinase activity of protealysin. Biochemistry (Mosc.), 2009, 74, 648-654.
[26]
Tsaplina, O.; Efremova, T.; Demidyuk, I.; Khaitlina, S. Filamentous actin is a substrate for protealysin, a metalloprotease of invasive Serratia proteamaculans. FEBS J., 2012, 279, 264-274.
[27]
Green, E.R.; Mecsas, J. Bacterial secretion systems: An overview. Microbiol. Spectr, 2016, 4, VMBF-0012-2015.
[28]
Murdoch, S.L.; Trunk, K.; English, G.; Fritsch, M.J.; Pourkarimi, E.; Coulthurst, S.J. The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors. J. Bacteriol., 2011, 193, 6057-6069.

Rights & Permissions Print Cite
Article Metrics
19
1
© 2024 Bentham Science Publishers | Privacy Policy