A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri strain 8111 is O- and N-glycosylated.

G.J. CAVALLERO; M. MALAMUD; A. C. CASABUONO; M. DE LOS Á. SERRADELL,; A. S. COUTO

JOURNAL OF PROTEOMICS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2017 vol. 162 p. 20 - 29

1874-3919

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacilluskefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometryanalysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequenceSSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decoratedwith galacturonic acid and another O-glycosylated site on peptide 471?476, witha Glc5?8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatographyanalysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. Significance: A detailed characterization of protein glycosylation is essential to establish the basis for understandingand investigating its biological role. It is known that S-layer proteins fromkefir-isolated L. kefiri strains are involvedin the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chainsin this proteinmay help to understand these important properties. Furthermore, this is the first description of Nglycosidic chains in S-layer glycoprotein from Lactobacillus spp.