Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin: History
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Subjects: Pharmacology & Pharmacy
Contributor:
Sofia Khaitlina
,
Ekaterina Bozhokina
,
Olga Tsaplina
,
Tatiana Efremova

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

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 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].

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 [10] 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 N-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 [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 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 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].

This entry is adapted from the peer-reviewed paper 10.3390/ijms21114025

References

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