Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

Książek, Mirosław; Mizgalska, Danuta; Enghild, Jan J.; Scavenius, Carsten; Thøgersen, Ida B.; Potempa, Jan

doi:10.1074/jbc.m114.601716

Cited by 44 publications

(85 citation statements)

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“…In comparison, previously reported purification yields per liter were 25 µg, 3 and 10 mg for serpins from Tannerella forsythia , Thermococcus kodakaraensis and Bifidobacterium longum [10, 14, 22]. Altogether these data indicate that the Siropin 1 and 2 are well expressed in E. coli and that the used purification procedure allowed the achievement of high yields of serpins.…”

Section: Resultssupporting

confidence: 53%

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Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases

et al. 2016

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BackgroundIn eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied.ResultsThe two genes encoding for putative serpins from the human gut bacterium Eubacterium sireaum, display low sequence identities. These genes were overexpressed and the encoded proteins, named Siropins, were purified. Activity studies demonstrated that both purified proteins inhibited serine proteases but surprisingly they preferentially inhibited two human serine proteases (Human Neutrophil Elastase and Proteinase3). The biochemical characterization of these Siropins revealed that Siropin 1 was the most active and stable at low pH values while Siropin 2 was more thermoactive and thermostable. Kinetic analysis allowed the determination of the stoichiometry of inhibition (SI) which was around 1 and of the association rate constants of 7.7 × 104 for the Human Neutrophil Elastase and 2.6 × 105 for the Proteinase3. Moreover, both Siropins displayed the ability to inhibit proteases usually present in fecal waters. Altogether our data indicate the high efficiency of Siropins and their probable involvement in the control of the overall intestine protease activity.ConclusionsHere we report the purification and the biochemical characterization of two novel serpins originated from Eubacterium sireaum, a human gastro-intestinal tract commensal bacteria. These proteins that we called Siropins, efficiently inhibited two human proteases reported to be associated with inflammatory bowel diseases. The determination of the biochemical properties of these enzymes revealed different temperature and pH behaviours that may reflect adaptation of this human commensal bacterium to different ecological environments. To the best of our knowledge, it is the first bacterial serpins showing an attractive inhibition of fecal proteases recovered from a mice group with chemically induced inflammation. Altogether our data highlight the interesting potential of Siropins, and serpins from the human gut microbiota in general, to be used as new alternative to face inflammatory diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0596-2) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 53%

“…2a, c, f and g), suggesting that both serpins from E. siraeum have a monomeric arrangement. Such organization is similar to those adopted by serpins from Thermococcus kodakaraensis , Tannerella forsythia, Pyrobaculum aerophilum and Thermobifida fusca [10, 12, 22, 23]. …”

Section: Resultsmentioning

confidence: 57%

Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases

et al. 2016

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“…Gels were stained with SimplyBlue™SafeStain (Novex) and washed in distilled water. Gel lanes were cut into sections and subjected to mass spectroscopy and follow up analysis, as described earlier65.…”

Section: Methodsmentioning

confidence: 99%

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Łasica

Goulas

Mizgalska

et al. 2016

Sci Rep

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Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

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“…Since the residue before the cleavage site directly interacts with the primary specificity pocket of a target protease, property of the P1 residue largely governs the rate and stoichiometry of inhibition. In some cases, one serpin ( e.g ., α1-protease inhibitor, α2-antiplasmin, miropin) inhibits several proteases with different specificity (Enghild et al, 1993; Ksiazek et al, 2015). These enzymes can cleave the inhibitor at a single or multiple peptide bonds in the loop, suggestive of an induced fit between the serpin and proteases.…”

Section: Discussionmentioning

confidence: 99%

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

Wang

Zhao

et al. 2017

Insect Biochemistry and Molecular Biology

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Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R366 at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R410 as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg366 by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg410 for trypsin-like proteases, Gly406 and Ala409 for the elastase and Thr404 for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0–50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13–containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.

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Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

Cited by 44 publications

References 49 publications

Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases

Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta

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