Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis

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 approa...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2005-12, Vol.5 (18), p.4808-4817
Main Authors: Pucciarelli, M. Graciela, Calvo, Enrique, Sabet, Christophe, Bierne, Hélène, Cossart, Pascale, García-del Portillo, Francisco
Format: Article
Language:English
Subjects:
2DnLC-MS/MS
Amino Acid Sequence
Aminoacyltransferases - metabolism
Analytical, structural and metabolic biochemistry
Bacterial Proteins - analysis
Bacterial Proteins - metabolism
Biochemistry, Molecular Biology
Biological and medical sciences
Cell Wall - chemistry
Chromatography, Liquid - methods
Cysteine Endopeptidases
Fundamental and applied biological sciences. Psychology
Life Sciences
Listeria
Listeria monocytogenes
Listeria monocytogenes - enzymology
Listeria monocytogenes - genetics
LPXTG
Mass Spectrometry - methods
Miscellaneous
Molecular Sequence Data
Nanotechnology - methods
Proteins
Proteomics - methods
Sortase
Substrate
Substrate Specificity
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Summary: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.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.200402075