title: A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.
abstract: Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90β, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands;
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.forename: Oleg I
authors.initials: OI
authors.lastname: Klychnikov
authors.forename: Dennis
authors.initials: D
authors.lastname: Bakker
authors.forename: Vincent J C
authors.initials: VJ
authors.lastname: van Winden
authors.forename: Nienke
authors.initials: N
authors.lastname: Ras
authors.forename: Arjan C
authors.initials: AC
authors.lastname: Kemp
authors.forename: Robert A
authors.initials: RA
authors.lastname: Cordfunke
authors.forename: Irina
authors.initials: I
authors.lastname: Dragan
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Ed J
authors.initials: EJ
authors.lastname: Kuijper
authors.forename: Jeroen
authors.initials: J
authors.lastname: Corver
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Hans C
authors.initials: HC
authors.lastname: van Leeuwen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D016360:Clostridioides difficile / Q000201:enzymology*; D003015:Clostridium Infections / Q000469:parasitology; D019143:Evolution, Molecular; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010802:Phylogeny; D011392:Proline / Q000378:metabolism*; D021382:Protein Sorting Signals; D020543:Proteome / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D018841:HSP90 Heat-Shock Proteins; C493165:HSP90AB1 protein, human; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D020543:Proteome; D011392:Proline; D045726:Metalloproteases