Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners

MALAMUD, MARIANO; CAVALLERO, GUSTAVO J.; CASABUONO, ADRIANA C.; LEPENIES, BERND; SERRADELL, MARÍA DE LOS ÁNGELES; COUTO, ALICIA S.

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2020 vol. 295 p. 14430 - 14444

0021-9258

The study of bacterial surface glycoproteins has become an expanding field of research in recent decades. S-layer proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle and its adjuvanticity depends on the integrity of its glycans. Herein, we analyze the impact of the glycosylation pattern on the recognition by C-type lectin receptors (CLRs) and the immunomodulatory effect of the L. kefiri SLPs on antigen presenting cells. Differences in the glycosylation of SLP-8348, SLP-8321 and SLP-5818 were detected. HPAEC-PAD performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs, however, some differences in the length of hexoses chains were observed. Regarding N-glycosylation, no signals were detected in SLP-8348 and SLP-8321. However, the presence of two N-glycosylation sites carrying complex N-glycans in SLP-5818 was proved by a site-specific analysis and a glycomic workflow of the permethylated glycans. Interestingly, as it was previously demonstrated for SLP-8348, SLP-8321 and SLP-5818 enhanced LPS-induced activation on both RAW264.7 macrophages and murine BMDCs regardless of the differences in their glycosylation patterns. Studies performed with BMDCs from CLR-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818 does not exert its effect in the absence of SignR3.