Fig. 2
Recognition of glycosylated LspA by SIGNR1. a O-linked glycan analysis of β-eliminated and permethylated G-LspA protein. Asterisk (*) indicates the contamination peak derived from reagents. b Glycosyl composition analysis of monosaccharides in the G-LspA sample. Trace levels of glucose were detected as a common contaminant derived from reagents. c Summary table showing the relative percentage of O-linked glycans from G-LspA. d LspA glycopeptides identified by glycoproteomics. The glycan composition and potential glycosylation sites (bolded) are shown. e Glycosyl linkage analysis of the O-glycans. Asterisks (*) indicate non-carbohydrate peaks. RT, retention time. f Binding of G-LspA to SIGNR1-hFc. Equal amounts of G-LspA and NG-LspA proteins were separated by SDS-PAGE and the specific interactions with SIGNR1-hFc were demonstrated, as no binding was detected in the presence of EDTA. g ELISA binding assays demonstrating G-LspA binding specificity with SIGNR1-hFc. The binding was abolished in the presence of EDTA, competitive zymosan, or blocking antibody to SIGNR1. h ELISA showing G-LspA did not bind to Dectin-1-hFc, as a control fusion protein. Zymosan served as a positive control. i Binding kinetics between G-LspA and SIGNR1-hFc. Various amounts of LspA proteins were coated on ELISA plates and incubated with SIGNR1-hFc (0.5 μg/ml). Binding was detected using HRP-conjugated anti-human IgG antibody. Kd, LspA concentration required to achieve a half-maximum binding with SIGNR1-hFc
