Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin: Comparison
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The entry reviews the discovery, properties, and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of

The article reviews the discovery, properties, and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of

Serratia grimesii

and protealysin of

Serratia proteamaculans

, characterized by both a highly specific “actinase” activity and their ability to stimulate bacterial invasion. Grimelysin cleaves the only one polypeptide bond Gly42-Val43 in actin. This bond is not cleaved by any other proteases and leads to a reversible loss of actin polymerization. Similar properties were characteristic for another bacterial protease, protealysin. These properties made grimelysin and protealysin a unique tool to study the functional properties of actin. Furthermore, bacteria

Serratia

spp. producing grimelysin/protealysin invade eukaryotic cells, and the recombinant

Escherichia coli

expressing the grimelysin or protealysins gene become invasive. Being an intracellular enzyme, grimelysin/protealysin can be delivered by bacteria to eukaryotic cells. These data indicate that the protease is a virulence factor, and actin can be a target for the protease upon its translocation into the host cell.

  • actin proteolysis
  • metalloproteinases
  • protease ECP 32
  • grimelysin
  • protealysin
  • bacterial invasion

1. Introduction

[1]Grimelysin (ECP 32)

, discovered, purified and initially characterized as protease ECP 32 [1][2][3], was later shown to be identical to grimelysin [4]. Therefore, the properties of the enzyme identified for ECP 32 could be applied to grimelysin. However, here we retain the name grimelysin (ECP 32) and ECP-cleaved actin to comply with the published data where the protease was named ECP 32. Grimelysin (ECP 32), purified from a bacterial extract using sequential chromatography steps, is a single 32 kDa polypeptide, whose

, discovered, purified and initially characterized as protease ECP 32 [6,7,8], was later shown to be identical to grimelysin [18]. Therefore, the properties of the enzyme identified for ECP 32 could be applied to grimelysin. However, here we retain the name grimelysin (ECP 32) and ECP-cleaved actin to comply with the published data where the protease was named ECP 32. Grimelysin (ECP 32), purified from a bacterial extract using sequential chromatography steps, is a single 32 kDa polypeptide, whose

N-terminal sequence was determined to be AKTSSAGVVIRDIFL [3]. The optimum of the protease activity was observed in the range of pH 7–8 when actin and melittin were used as substrates [3][5]. The proteolytic activity increased with increasing ionic strength: in 50–100 mM NaCl the activity of grimelysin (ECP 32) towards melittin was shown to be nearly twice higher than in a low ionic strength solution [5][6]. It was also enhanced in the presence of millimolar ATP concentrations, though hydrolysis of melittin was not accompanied by ATP hydrolysis at a rate comparable with the cleavage rate. This implies that protease grimelysin (ECP 32) is not an ATP-dependent enzyme [5], which is important for the experiments involving actin because actin contains ATP as a tightly-bound nucleotide. The protease activity is inhibited by EDTA, EGTA, o-phenanthroline and zincone, and the EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions [7][8]. Based on these data, grimelysin (ECP 32) was classified as a neutral metalloproteinase (EC 3.4.24) [3].

-terminal sequence was determined to be AKTSSAGVVIRDIFL [8]. The optimum of the protease activity was observed in the range of pH 7–8 when actin and melittin were used as substrates [8,23]. The proteolytic activity increased with increasing ionic strength: in 50–100 mM NaCl the activity of grimelysin (ECP 32) towards melittin was shown to be nearly twice higher than in a low ionic strength solution [23,24]. It was also enhanced in the presence of millimolar ATP concentrations, though hydrolysis of melittin was not accompanied by ATP hydrolysis at a rate comparable with the cleavage rate. This implies that protease grimelysin (ECP 32) is not an ATP-dependent enzyme [23], which is important for the experiments involving actin because actin contains ATP as a tightly-bound nucleotide. The protease activity is inhibited by EDTA, EGTA, o-phenanthroline and zincone, and the EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions [2,3]. Based on these data, grimelysin (ECP 32) was classified as a neutral metalloproteinase (EC 3.4.24) [8].

Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 [9] was observed at enzyme: substrate mass ratios of 1:25 to 1:3000 [3]. Two more sites, between Ala-29 and Val-30 and between Ser-33 and Ile-34, were cleaved by ECP 32 in heat- or EDTA-inactivated actin, apparently due to conformational changes around residues 28–34 buried in intact actin [3]. Besides actin, only melittin [10][11], histone H5, bacterial DNA-binding protein HU and chaperone DnaK [12] were found to be protease substrates. In agreement with this high substrate specificity, ECP 32 did not hydrolyze tropomyosin, troponin, α-actinin, casein, histone H2B, ovalbumin, bovine serum IgG, bovine serum albumin, bovine pancreatic ribonuclease A, trypsin, human heat shock protein HSP70, chicken egg lysozyme [2] insulin [6], DNAse I [13][14], gelsolin [15] and profilin [16]. The amino acid residues recognized by grimelysin (ECP 32) in actin and melittin are hydrophobic. This specificity is characteristic for thermolysin-like metalloproteinases [17]. However, high specificity of the enzyme seems to be determined predominantly by conformation at the actin cleavage site rather than its primary structure.

Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 [10] was observed at enzyme: substrate mass ratios of 1:25 to 1:3000 [8]. Two more sites, between Ala-29 and Val-30 and between Ser-33 and Ile-34, were cleaved by ECP 32 in heat- or EDTA-inactivated actin, apparently due to conformational changes around residues 28–34 buried in intact actin [8]. Besides actin, only melittin [18,19], histone H5, bacterial DNA-binding protein HU and chaperone DnaK [25] were found to be protease substrates. In agreement with this high substrate specificity, ECP 32 did not hydrolyze tropomyosin, troponin, α-actinin, casein, histone H2B, ovalbumin, bovine serum IgG, bovine serum albumin, bovine pancreatic ribonuclease A, trypsin, human heat shock protein HSP70, chicken egg lysozyme [7], insulin [24], DNAse I [9,13], gelsolin [14] and profilin [26]. The amino acid residues recognized by grimelysin (ECP 32) in actin and melittin are hydrophobic. This specificity is characteristic for thermolysin-like metalloproteinases [27]. However, high specificity of the enzyme seems to be determined predominantly by conformation at the actin cleavage site rather than its primary structure.

2.        Grimelysin

Grimelysin was obtained as a recombinant protein. This has been achieved by cloning the putative gene encoding grimelysin in

was obtained as a recombinant protein. This has been achieved by cloning the putative gene encoding grimelysin in

S. grimesii

A2 and in the reference

S. grimesii 30063 [10] using published protealysin sequences identified in

30063 [18] using published protealysin sequences identified in

S. proteamaculans [11]. Grimelysin shared all properties characteristic for ECP 32 including a molecular weight of 32 kDa, an

[19]. Grimelysin shared all properties characteristic for ECP 32 including a molecular weight of 32 kDa, an

N-terminal 14 amino acid sequence, optimum activity in the range of pH 7–8 and inhibition with o-phenanthroline and EGTA [10].

-terminal 14 amino acid sequence, optimum activity in the range of pH 7–8 and inhibition with o-phenanthroline and EGTA [18].

3.

      

Protealysin

Protealysin is a neutral zink-containing metalloprotease of

is a neutral zink-containing metalloprotease of

Serratia proteamaculans

. The protealysin gene was cloned from a genomic library of

S. proteamaculans

strain 94 isolated from spoiled meat. This protein was expressed in

Escherichia coli and purified as described earlier [11]. Similarly to other thermolysin-like proteases [17][18], protealysin is synthesized as a precursor containing a propeptide of about 50 amino acids that is removed during formation of mature active protein [19]. The propeptide is much shorter than the propeptides of the thermolysin-like proteases and has no significant structural similarity to the propeptides of most thermolysin-like proteases [20][21]. A similar propeptide of 50 amino acids was also detected in the primary structure of the recombinant grimelysin. According to SDS-electrophoresis, recombinant proteins with or without propeptide had an apparent molecular weight of 37 and 32 kDa, respectively [11].

and purified as described earlier [19]. Similarly to other thermolysin-like proteases [27,28], protealysin is synthesized as a precursor containing a propeptide of about 50 amino acids that is removed during formation of mature active protein [29]. The propeptide is much shorter than the propeptides of the thermolysin-like proteases and has no significant structural similarity to the propeptides of most thermolysin-like proteases [30,31,32]. A similar propeptide of 50 amino acids was also detected in the primary structure of the recombinant grimelysin. According to SDS-electrophoresis, recombinant proteins with or without propeptide had an apparent molecular weight of 37 and 32 kDa, respectively [19].

The molecular weight of the active recombinant protealysin 32 kDa and the

 The molecular weight of the active recombinant protealysin 32 kDa and the

N-terminal amino acid sequence AKTSTGGEVI are identical to those of grimelysin [3][11]. The optimal pH for azocasein hydrolysis is 7, and protealysin is completely inhibited by

-terminal amino acid sequence AKTSTGGEVI are identical to those of grimelysin [8,19]. The optimal pH for azocasein hydrolysis is 7, and protealysin is completely inhibited by

o-phenanthroline [11], i.e., has the same properties as grimelysin [3][4]. Protealysin and grimelysin (ECP 32) are also similar in their unique property of being able to digest actin specifically [3][9][13][22][23].

-phenanthroline [19], i.e., has the same properties as grimelysin [8,18]. Protealysin and grimelysin (ECP 32) are also similar in their unique property of being able to digest actin specifically [8,9,10,22,33].
 
 
 
  • Mantulenko, V.B.; Khaitlina, S.Y.; Sheludko, N.S. High molecular weight proteolysis-resistant actin fragment. Biochemistry 1983, 48, 69–74. [Google Scholar]
  • Usmanova, A.M.; Khaitlina, S.Y. A specific actin-digesting protease from the bacterial strain E.coli A2. Biochemistry 1989, 54, 1074–1079. [Google Scholar]
  • Matveyev, V.V.; Usmanova, A.M.; Morozova, A.B.; Khaitlina, S.Y. Purification and characterization of the proteinase ECP-32 from Escherichia coli A2 strain. Biochim. Biophys. Acta 1996, 1296, 55–62. [Google Scholar] [CrossRef]
  • Khaitlina, S.Y.; Smirnova, T.D.; Usmanova, A.M. Limited proteolysis of actin by a specific bacterial protease. FEBS Lett. 1988, 28, 72–74. [Google Scholar] [CrossRef]
  • Khaitlina, S.Y.; Collins, J.H.; Kusnetsova, I.M.; Pershina, V.P.; Synakevich, I.G.; Turoverov, K.K.; Usmanova, A.M. Physico-chemical properties of actin cleaved with bacterial protease from E. coli A2 strain. FEBS Lett. 1991, 279, 49–51. [Google Scholar] [CrossRef]
  • Kabsch, W.; Mannherz, H.G.; Suck, D.; Pai, E.F.; Holmes, K.C. Atomic structure of the actin:DNase I complex. Nature 1990, 347, 37–44. [Google Scholar] [CrossRef] [PubMed]
  • Holmes, K.C.; Popp, D.; Gebhard, W.; Kabsch, W. Atomic model of the actin filament. Nature 1990, 347, 44–49. [Google Scholar] [CrossRef] [PubMed]
  • Khaitlina, S.Y.; Moraczewska, J.; Strzelecka-Golaszewska, H. The actin-actin interactions involving the N-terminal portion of the DNase I-binding loop are crucial for stabilization of the actin filament. Eur. J. Biochem. 1993, 218, 911–920. [Google Scholar] [CrossRef] [PubMed]
  • Khaitlina, S.; Hinssen, H. Conformational changes in actin Induced by Its Interaction with gelsolin. Biophys. J. 1997, 73, 929–937. [Google Scholar] [CrossRef]
  • Khaitlina, S.Y.; Strzelecka-Golaszewska, H. Role of the DNase-I-binding loop in dynamic properties of actin filament. Biophys. J. 2002, 82, 321–334. [Google Scholar] [CrossRef]
  • Moraczewska, J.; Gruszczynska-Biegala, J.; Redowicz, M.J.; Khaitlina, S.; Strzelecka-Golaszewska, H. The DNase-I binding loop of actin may play a role in the regulation of actin-myosin interaction by tropomyosin/troponin. J. Biol. Chem. 2004, 279, 31197–31204. [Google Scholar] [CrossRef]
  • Khaitlina, S.; Tsaplina, O.; Hinssen, H. Cooperative effects of tropomyosin on the dynamics of the actin filament. Febs Lett. 2017, 591, 1884–1891. [Google Scholar] [CrossRef]
  • Bozhokina, E.; Khaitlina, S.; Adam, T. Grimelysin, a novel metalloprotease from Serratia grimesii, is similar to ECP 32. Biochem Biophys Res Commun. 2006, 367, 888–892. [Google Scholar] [CrossRef]
  • 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. [Google Scholar] [CrossRef]
  • Efremova, T.; Ender, N.; Brudnaja, M.; Komissarchik, Y.; Khaitlina, S. Specific invasion of transformed cells by Escherichia coli A2 strain. Cell Biol. Intern. 2001, 25, 557–561. [Google Scholar] [CrossRef]
  • 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. [Google Scholar] [CrossRef] [PubMed]
  • 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 2009, 74, 648–654. [Google Scholar] [CrossRef] [PubMed]
  • Kazanina, G.A.; Mirgorodskaia, E.P.; Mirgorodskaia, O.A.; Khaitlina, S.Y. ECP 32 proteinase: Characteristics of the enzyme, study of specificity. Bioorg. Khim. 1995, 21, 761–766. [Google Scholar] [PubMed]
  • Mirgorodskaya, O.; Kazanina, G.; Mirgorodskaya, E.; Matveyev, V.; Thiede, B.; Khaitlina, S.Y. Proteolytic cleavage of mellitin with the actin-digesting protease. Protein Pept. Lett. 1996, 3, 81–88. [Google Scholar]
  • Morozova, A.V.; Khaitlina, S.Y.; Malinin, A.Y. Heat Shock Protein DnaK—Substrate of actin-specific bacterial protease ECP 32. Biochemistry 2011, 76, 455–461. [Google Scholar] [CrossRef]
  • Khaitlina, S.; Lindberg, U. Dissociation of profilactin as a two-step process. J. Muscle Res. Cell Motil. 1995, 16, 188–189. [Google Scholar]
  • Rawlings, N.D.; Burrett, A.J. Evolutionary families of metallopeptidases. Methods Enzym. 1995, 248, 183–228. [Google Scholar] [PubMed]
  • Shinde, U.; Inouye, M. Intramolecular chaperones: Polypeptide extensions that modulate protein folding. Semin. Cell Dev. Biol. 2000, 11, 35–44. [Google Scholar] [CrossRef]
  • Gromova, T.Y.; Demidyuk, I.V.; Kozlovskiy, V.I.; Kuranova, I.P.; Kostrov, S.V. Processing of protealysin precursor. Biochimie 2009, 91, 639–645. [Google Scholar] [CrossRef]
  • 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. [Google Scholar] [CrossRef]
  • 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. [Google Scholar] [CrossRef] [PubMed]
  • Demidyuk, I.V.; Shubin, A.V.; Gasanov, E.V.; Kostrov, S.V. Propeptides as modulators of functional activity of proteases. Biomol. Concepts 2010, 1, 305–322. [Google Scholar] [CrossRef]
  • 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. [Google Scholar] [CrossRef] [PubMed]

References

  1. Mantulenko, V.B.; Khaitlina, S.Y..; Sheludko, N.S; High molecular weight proteolysis-resistant actin fragment. Sofia Khaitlina , , Ekaterina Bozhokina, , Olga Tsaplina Tatiana Efremova; Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin as Virulence Factors Contributing to the Invasive Activities of Serratia. Int.J.Mol.Biochemistry 1983, 48, 69–74.l. 2020, 21, E4025., 10.3390/ijms21114025 .
  2. Usmanova, A.M.; Khaitlina, S.Y; A specific actin-digesting protease from the bacterial strain E.coli A2. Biochemistry 1989, 54, 1074–1079.
  3. Vladimir V. Matveyev; Aislu M. Usmanova; Alevtina V. Morozova; John H. Collins; Sofia Yu. Khaitlina; Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1996, 1296, 55-62, 10.1016/0167-4838(96)00053-2.
  4. Bozhokina, E.; Khaitlina, S.; Adam, T; Grimelysin, a novel metalloprotease from Serratia grimesii, is similar to ECP 32. Biochem Biophys Res Commun. 2006, 367, 888–892.
  5. G A Kazanina; E P Mirgorodskaia; O A Mirgorodskaia; S Iu Khaĭtlina; [ECP 32 proteinase: characteristics of the enzyme, study of specificity].. Биоорганическая химия 1995, 21, 761–766.
  6. Mirgorodskaya, O.; Kazanina, G.; Mirgorodskaya, E.; Matveyev, V.; Thiede, B.; Khaitlina, S.Y; Proteolytic cleavage of mellitin with the actin-digesting protease. Protein Pept. Lett. 1996, 3, 81–88.
  7. Pamela Schnupf; Philippe J. Sansonetti; Shigella Pathogenesis: New Insights through Advanced Methodologies. Microbiology Spectrum 2019, 7, 15-39, 10.1128/microbiolspec.bai-0023-2019.
  8. Zhiwei Huang; Sarah E Sutton; Adam J Wallenfang; Robert C Orchard; Xiaojing Wu; Yingcai Feng; Jijie Chai; Neal M Alto; Structural insights into host GTPase isoform selection by a family of bacterial GEF mimics. Nature Structural & Molecular Biology 2009, 16, 853-860, 10.1038/nsmb.1647.
  9. S.Yu. Khaitlina; J.H. Collins; I. M. Kuznetsova; V.P. Pershina; I.G. Synakevich; K.K. Turoverov; A.M. Usmanova; Physico-chemical properties of actin cleaved with bacterial protease from E. coli A2 strain. FEBS Letters 1991, 279, 49-51, 10.1016/0014-5793(91)80247-z.
  10. Ekaterina Bozhokina; Sofia Khaitlina; Thomas Adam; Grimelysin, a novel metalloprotease from Serratia grimesii, is similar to ECP32. Biochemical and Biophysical Research Communications 2008, 367, 888-892, 10.1016/j.bbrc.2008.01.003.
  11. Ilya Demidyuk; Alexander Kalashnikov; Tatiana Yu. Gromova; Eugene Gasanov; Dina R. Safina; Maria V. Zabolotskaya; G. N. Rudenskaya; Sergey V. Kostrov; 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 Expression and Purification 2006, 47, 551-561, 10.1016/j.pep.2005.12.005.
  12. A. V. Morozova; S. Yu. Khaitlina; A. Yu. Malinin; Heat shock protein DnaK — Substrate of actin-specific bacterial protease ECP32. Biochemistry (Moscow) 2011, 76, 455-461, 10.1134/s0006297911040080.
  13. S.Yu. Khaitlina; T.D. Smirnova; A.M. Usmanova; Limited proteolysis of actin by a specific bacterial protease. FEBS Letters 1988, 228, 72-74, 10.1016/0014-5793(88)80610-0.
  14. Khaitlina, S.Y..; Moraczewska, J.; Strzelecka-Golaszewska, H; The actin-actin interactions involving the N-terminal portion of the DNase I-binding loop are crucial for stabilization of the actin filament. Eur. J. Biochem. 1993, 218, 911–920.
  15. S. Khaitlina; H. Hinssen; Conformational changes in actin induced by its interaction with gelsolin. Biophysical Journal 1997, 73, 929-937, 10.1016/s0006-3495(97)78125-6.
  16. Khaitlina, S.; Lindberg, U; Dissociation of profilactin as a two-step process. J. Muscle Res. Cell Motil. 1995, 16, 188–189.
  17. N D Rawlings; A J Barrett; Evolutionary families of metallopeptidases. Part B: Numerical Computer Methods 1995, 248, 183–228.
  18. Ujwal Shinde; Masayori Inouye; Intramolecular chaperones: polypeptide extensions that modulate protein folding. Seminars in Cell & Developmental Biology 2000, 11, 35-44, 10.1006/scdb.1999.0349.
  19. Tania Yu. Gromova; Ilya Demidyuk; Viatcheslav Kozlovskiy; Inna P. Kuranova; Sergei Kostrov; Processing of protealysin precursor. Biochimie 2009, 91, 639-645, 10.1016/j.biochi.2009.03.008.
  20. Ilya Demidyuk; Eugene Gasanov; Dina R. Safina; Sergey V. Kostrov; Structural Organization of Precursors of Thermolysin-like Proteinases. The Protein Journal 2008, 27, 343-354, 10.1007/s10930-008-9143-2.
  21. 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.
  22. Olga Tsaplina; T. N. Efremova; L. V. Kever; Ya. Yu. Komissarchik; Ilya Demidyuk; S. V. Kostrov; S. Yu. Khaitlina; Probing for actinase activity of protealysin. Biochemistry (Moscow) 2009, 74, 648-654, 10.1134/s0006297909060091.
  23. Olga Tsaplina; Tatiana Efremova; Ilya Demidyuk; Sofia Khaitlina; Filamentous actin is a substrate for protealysin, a metalloprotease of invasive Serratia proteamaculans. The FEBS Journal 2011, 279, 264-274, 10.1111/j.1742-4658.2011.08420.x.
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