Abstract
Understanding blood group antigen binding preferences for C-type lectin receptors holds promise for modulating immune responses, since several Gram-negative bacteria express blood group antigens as molecular mimicry to evade immune responses. Herein, we report the synthesis of ABO blood group antigen active tri and disaccharides to investigate the binding specificity with various C-type lectin receptors using glycan microarray. The results of binding preferences show that distinct glycosylation on the galactose and fucose motifs are key for C-type lectin receptor binding and that these interactions occur in a Ca2+-dependent fashion.
Highlights
The donor 8 was synthesized from a known thiogalactoside donor 15, which was synthesized from D-galactose in 4 steps, as described22
5′-GAATTCGTCCAAGGTCCCCAGCTCCAT-3′ 5′-CCATGGACGCAGGAGGGGGGTTTGGGGT-3′ 5′-GAATTCCATGCAACTGAAGGCTGAAG-3′ 5′-AGATCTTTTGGTGGTGCATGATGAGG-3′ 5′-CCATGGGGCAGAACTTACAGCCACAT-3′ 5′-AGATCTGTCCAGAGGACTTATTTCTG-3′ 5′-CCAGTTAAGGAGGGACCTAGGCAC-3′ 5′-AGCTCTCCTTGGCCAGCTTCATC-3′. These results provide the basis for understanding of how pathogens may target C-type lectin receptor (CLR) by molecular mimicry to evade immune responses
We have synthesized a series of blood group antigens and immobilized them on glycan microarray slides to determine C-type lectin receptor binding preferences
Summary
The synthetic [1,2,3,4] were printed onto epoxide-functionalized microarray slides at 50 μM in replicates of four, as described in the experimental section[24,25]. CLR-hFc fusion proteins were incubated on the slide at 20 ng μl−1 (DC-SIGN, SIGNR3, Mincle and MGL-1)26,27 in optimized conditions of 50 mM HEPES buffer with 5 mM CaCl2, 5 mM MgCl2, 0.005% Tween-20 and 1% ovalbumin, followed by the secondary antibody (Cy3-anti-human IgG). Slides were scanned and the binding was determined by the fluorescence intensity, as described in the experimental section (Fig. 4).
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