title: Substrate cleavage profiling suggests a distinct function of Bacteroides fragilis metalloproteinases (fragilysin and metalloproteinase II) at the microbiome-inflammation-cancer interface.
abstract: Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.
authors.affiliation: From the Sanford-Burnham Medical Research Institute, La Jolla, California 92037 and.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Shiryaev
authors.forename: Albert G
authors.initials: AG
authors.lastname: Remacle
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Chernov
authors.forename: Vladislav S
authors.initials: VS
authors.lastname: Golubkov
authors.forename: Khatereh
authors.initials: K
authors.lastname: Motamedchaboki
authors.forename: Norihito
authors.initials: N
authors.lastname: Muranaka
authors.forename: Corey M
authors.initials: CM
authors.lastname: Dambacher
authors.forename: Petr
authors.initials: P
authors.lastname: Capek
authors.forename: Muskan
authors.initials: M
authors.lastname: Kukreja
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kozlov
authors.forename: Manuel
authors.initials: M
authors.lastname: Perucho
authors.forename: Piotr
authors.initials: P
authors.lastname: Cieplak
authors.forename: Alex Y
authors.initials: AY
authors.lastname: Strongin
mesh_terms: D000595:Amino Acid Sequence; D001441:Bacteroides fragilis / Q000235:genetics* / Q000472:pathogenicity; D044404:Genomic Islands / Q000235:genetics; D006801:Humans; D007249:Inflammation / Q000235:genetics*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D064307:Microbiota; D009369:Neoplasms / Q000235:genetics / Q000473:pathology; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D013379:Substrate Specificity
chemical_list: D043484:Proprotein Convertases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; C091198:fragilysin
keywords: Bacterial Toxins; Bacteroides; Enzymes; Fragilysin; Metalloprotease; Microbial Pathogenesis; Toxins