Difference in fine specificity to polysaccharides of Candida albicans mannoprotein between mouse SIGNR1 and human DC-SIGN

Abstract

C-type lectin SIGNR1 directly recognizes Candida albicans and zymosan and has been considered to share properties of polysaccharide recognition with human DC-SIGN (hDC-SIGN). However, the precise specificity of SIGNR1 and the difference from that of hDC-SIGN remain to be elucidated. We prepared soluble forms of SIGNR1 and hDC-SIGN and conducted experiments to examine their respective specificities. Soluble SIGNR1 (sSIGNR1) bound several types of live C. albicans clinical isolate strains in an EDTA-sensitive manner. Inhibition analyses of sSIGNR1 binding by glycans from various yeast strains demonstrated that SIGNR1 preferentially recognizes N-glycan α-mannose side chains in Candida mannoproteins, as reported in hDC-SIGN. Unlike shDC-SIGN, however, sSIGNR1 recognized not only Saccharomyces cerevisiae, but also C. albicans J-1012 glycan, even after α-mannosidase treatment that leaves only β1,2-mannose-capped α-mannose side chains. In addition, glycomicroarray analyses showed that sSIGNR1 binds mannans from C. albicans and S. cerevisiae but does not recognize Lewis(a/b/x/y) antigen polysaccharides as in shDC-SIGN. Consistent with these results, RAW264.7 cells expressing hDC-SIGN in which the carbohydrate recognition domain (CRD) was replaced with that of SIGNR1 (RAW-chimera) produced comparable amounts of interleukin 10 (IL-10) in response to glycans from C. albicans and S. cerevisiae, but those expressing hDC-SIGN produced less IL-10 in response to S. cerevisiae than C. albicans. Furthermore, RAW-hDC-SIGN cells remarkably reduced IL-10 production after α-mannosidase treatment compared with RAW-chimera cells. These results indicate that SIGNR1 recognizes C. albicans/yeast through a specificity partly distinct from that of its homologue hDC-SIGN.

Fig 1

sSIGNR1 binds various types of yeast strains. (A) Structural diagrams of N-glycans used in this study. The structures of N-glycans of C. albicans J-1012 (25), C. albicans NIH B-792 (22), C. stellatoidea (24), C. parapsilosis (22), and C. lusitaniae (23) are adopted from our structural analyses using nuclear magnetic resonance (NMR). The structures of S. cerevisiae wild type, S. cerevisiae 4484-24D-1 (mnn1/mnn4), and S. cerevisiae X2180-1-A-5 (mnn2) are based on previous reports (1, 21). Side chains that are digested by treatment with α-mannosidase in C. albicans J-1012 N-glycan are shaded. The side chain sequence is not specified. (B) Binding of sSIGNR1 to Candida and S. cerevisiae strains. PE–Strep-Tactin alone (−) or PE-sSIGNR1 (+) was incubated with the indicated live yeast strains with or without EDTA (25 mM). (C) Inhibition of sSIGNR1 binding to C. albicans J-1012 by glycans purified from the C. albicans and S. cerevisiae strains indicated. sSIGNR1 was preincubated with 50 μg/ml of glycans before mixing with microbes. Glycogen was used as a negative control. Inhibition is indicated as the percent decrease of fluorescence intensity in experimental groups compared with the control without inhibitor. The results are shown as the means ± SD of triplicate assays. *, P < 0.05 (solid line) by Tukey's multiple-range test. The gray lines indicate no significant differences.

Fig 2

Recognition of α-mannose side chains in N-glycan by sSIGNR1. (A) Inhibition analysis by lectin ELISA. Binding of sSIGNR1 to microtiter plates coated with C. albicans J-1012 glycan was analyzed in the presence of glycans (25 μg/ml) purified from various types of yeast strains. Blocking activities of inhibitors are shown as the percent inhibition of sSIGNR1 binding. (B) Titration of inhibitory activity of glycans from the indicated yeast strains for sSIGNR1 binding by lectin ELISA. Half of maximal inhibition activity was indicated by the dashed line. (C) Inhibition of FITC binding to RAW-SIGNR1 cells by glycans. Transfectants were incubated with graded doses of glycans as in panel B prior to FITC-dextran. The results are shown as percent inhibition (means ± SD of triplicate assays). *, P < 0.05 (solid lines) by Tukey's multiple-range test. The gray lines indicate no significant differences.

Fig 3

Binding of shDC-SIGN to microbes and inhibition of shDC-SIGN binding by glycans. (A) Inhibition analysis of shDC-SIGN binding by monosaccharides (50 mM) and glycan from C. albicans J-1012 using lectin ELISA. (B) Binding of shDC-SIGN to yeast strains was analyzed as in Fig. 1B. (C) Inhibition analysis using glycans from various yeast strains as in Fig. 2A. (D) Inhibition assay performed in the presence of graded doses of glycans from the indicated yeast strains. Half of maximal inhibition activity was indicated by the dashed line. The results are shown as the means ± SD of triplicate assays. *, P < 0.05 (solid lines) by Tukey's multiple-range test. The gray lines indicate no significant differences.

Fig 4

Glycomicroarray analysis of sSIGNR1. (A) Layout of the glycomicroarray. (B) Results of glycomicroarray analyses. Binding of soluble SIGNR1/Alexa 647–anti-SIGNR1 MAb immune complex to the array was performed in the absence (left) or presence (right) of 10 mM EDTA and detected by an evanescent-field fluorescence-assisted scanner. (C) Data analyzed with the Array Pro analyzer ver. 4.5. (D) Glycoarray analysis was performed using an immune complex of soluble hDC-SIGN/Alexa 555–anti-hDC-SIGN MAb as for sSIGNR1.

Fig 5

IL-10 production of RAW264.7 transfectants upon stimulation with glycan on a plastic plate. (A) RAW-control, RAW–hDC-SIGN, and RAW-chimera cells were analyzed by flow cytometry using polyclonal anti-hDC-SIGN Ab and anti-SIGNR1 MAb (22D1) specific for the SIGNR1 CRD. (B) The transfectants (5 × 10⁴ cells) were cultured on plates precoated with C. albicans J-1012 and S. cerevisiae X2180-1A (WT) glycan in the presence or absence of LPS (100 ng/ml). After 24 h, IL-10 in the supernatants was analyzed (top). IL-10 production against S. cerevisiae glycan is shown as a percentage of that against C. albicans glycan (bottom). (C) IL-10 production after stimulation with native and α-mannosidase-treated C. albicans J-1012 glycan was analyzed as in panel B. IL-10 production against the treated glycan is shown as a percentage of that against the native glycan. **, P = 0.0067, and ***, P = 0.0001 by Student's t test.