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Rinaldi, Simon and Brennan, Kathryn M. and Goodyear, Carl S. and O'Leary, Colin and Schiavo, Giampietro and Crocker, Paul R. and Willison, Hugh J.;
Analysis of lectin binding to glycolipid complexes using combinatorial glycoarrays
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Glycolipids are major components of the plasma membrane, interacting with themselves, other lipids, and proteins to form an array of heterogeneous domains with diverse biological properties. Considerable effort has been focused on identifying protein binding partners for glycolipids and the glycan specificity for these interactions, largely achieved through assessing interactions between proteins and homogenous, single species glycolipid preparations. This approach risks overlooking both the enhancing and attenuating roles of heterogeneous glycolipid complexes in modulating lectin binding. Here we report a simple method for assessing lectin-glycolipid interactions. An automatic thin-layer chromatography sampler is employed to create easily reproducible arrays of glycolipids and their heterodimeric complexes immobilized on a synthetic polyvinyldifluoride membrane. This array can then be probed with much smaller quantities of reagents than would be required using existing techniques such as ELISA and thin-layer chromatography with immuno-overlay. Using this protocol, we have established that the binding of bacterial toxins, lectins, and antibodies can each be attenuated, enhanced, or unaffected in the presence of glycolipid complexes, as compared with individual, isolated glycolipids. These findings underpin the wide-ranging influence and importance of glycolipid-glycolipid cis interactions when the nature of protein-carbohydrate recognition events is being assessed.

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