title: Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis.
abstract: Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria by cleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif is LPXTG, which is cleaved between the T and G residues. In this study, a non-gel proteomic approach was used to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA and SrtB. Material containing peptidoglycan and strongly associated proteins was purified from sortase-defective mutants, digested with trypsin, and the resulting peptide mixture analysed by two-dimensional nano-liquid chromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycan metabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surface proteins, Lmo2185 and Lmo2186, were identified only when SrtB was active. The analysis of the peptides identified in these proteins suggests that SrtB of L. monocytogenes may recognize two different sorting motifs, NXZTN and NPKXZ. Taken together, these data demonstrate that non-gel proteomics is a powerful technique to rapidly identify sortase substrates and to gain insights on potential sorting motifs.
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain.
authors.forename: M Graciela
authors.initials: MG
authors.lastname: Pucciarelli
authors.forename: Enrique
authors.initials: E
authors.lastname: Calvo
authors.forename: Christophe
authors.initials: C
authors.lastname: Sabet
authors.forename: Hélène
authors.initials: H
authors.lastname: Bierne
authors.forename: Pascale
authors.initials: P
authors.lastname: Cossart
authors.forename: Francisco
authors.initials: F
authors.lastname: García-del Portillo
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D003546:Cysteine Endopeptidases; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases