Mass Spectrometry Analysis of the Extracellular Peptidome of Lactococcus lactis: Lines of Evidence for the Coexistence of Extracellular Protein Hydrolysis and Intracellular Peptide Excretion

• Published in: Journal of proteome research 2016-09, Vol.15 (9), p.3214-3224
• Main Authors: Guillot, Alain, Boulay, Mylène, Chambellon, Émilie, Gitton, Christophe, Monnet, Véronique, Juillard, Vincent
• Format: Article
• Language: English
• Subjects: Amino Acids, Branched-Chain - metabolism; Bacterial Proteins - metabolism; Cell Membrane - enzymology; Cytoplasm - enzymology; Life Sciences; Mass Spectrometry; Peptide Hydrolases - metabolism; Peptides - analysis; Peptides - metabolism; Proteolysis; Proteomics - methods; Serine Endopeptidases - metabolism
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.6b00424

We report here the use of a peptidomic approach to revisit the extracellular proteolysis of Lactococcus lactis. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain L. lactis IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins.