Abstract
Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.
Highlights
Surface layers (S-layers) are planar arrays of proteinaceus or glycoproteinaceus subunits ranging in molecular mass (Mr) from 40 to 200 kDa which can be aligned in unit cells of different symmetries on the outermost surface of many prokaryotic microorganisms
In order to characterize the immunoreactivity of surface proteins from different L. kefir strains, we obtained monoclonal antibodies (Mabs) against S-layer proteins from L. kefir CIDCA 8348
Two hybridomas secreting specific antibodies (Mabs 1F8 and 5F8) were selected. Both Mabs showed specificity at a species level in an indirect enzyme-linked immunosorbent assay (ELISA), since they did not react with S-layer proteins from L. brevis JCM 1059 and ATCC 8287
Summary
Surface layers (S-layers) are planar arrays of proteinaceus or glycoproteinaceus subunits ranging in molecular mass (Mr) from 40 to 200 kDa which can be aligned in unit cells of different symmetries on the outermost surface of many prokaryotic microorganisms. S-layer proteins have shown to be related with anti-pathogenic activity of some probiotic bacteria (Horie et al 2002; Johnson-Henry et al 2007; Golowczyc et al 2007). Presence of S-layer has been demonstrated in many species of genus Lactobacillus, but, to date, only 21 S-layer protein encoding genes have been cloned and sequenced, showing an overall lack of identity between different species. Glycosylation is commonly found in S-layer proteins, most of S-layers from lactobacilli appear to be non-glycosylated (AvallJaaskelainen and Palva 2005), and to date a detailed glycan structure has been reported only for S-layer from Lactobacillus buchneri (Moschl et al 1993)
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