Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin: Comparison
Please note this is a comparison between Version 2 by Rita Xu and Version 1 by Sofia Khaitlina.

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]
Grimelysin (ECP 32), 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

, 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

N-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].

-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].

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.

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.

       
Grimelysin

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 [18] using published protealysin sequences identified in

30063 [10] using published protealysin sequences identified in

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

[11]. 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 [18].

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

      
Protealysin

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 [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].

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].

 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 [8,19]. 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 [3][11]. The optimal pH for azocasein hydrolysis is 7, and protealysin is completely inhibited by

o-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]

-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].

References

  1. 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.Mantulenko, V.B.; Khaitlina, S.Y..; Sheludko, N.S; High molecular weight proteolysis-resistant actin fragment. Biol. 2020, 21, E4025., 10.3390/ijms21114025 .chemistry 1983, 48, 69–74.
  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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