Analysis of lectin binding to glycolipid complexes using combinatorial glycoarrays

Abstract

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 polyvinyl-difluoride 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.

Fig. 1

Row and column headings reveal the complex at each location. ‘X's represent negative controls (methanol only) and act as a line of symmetry for duplicate spots within the same membrane. (A) Siglec-7-Fc reacts with GD3 in isolation, yet the signal intensity for most complexes of GD3 is either abolished or much reduced (“complex attenuated”). (B) TeNT H_C does not bind to the single gangliosides GD3, GM1, GD1a or GT1a, yet reacts strongly with heterodimers of these gangliosides containing GD3 (“complex enhanced”). (C) Quantification of the modulatory effects of GD3 series complexes on siglec-7 and TeNT H_C binding. Asterisks denote complexes displaying binding levels significantly different from the sum of the two individual components. Complex modulated binding was defined as the sum of intensity readings for the single gangliosides (to a maximum of 100%) subtracted from the signal generated by the complex being significantly different to zero, shown by the 99.9% confidence interval for this difference failing to cross zero. Complexes fitting this definition, along with the magnitude of the effect, are plotted for Siglec-7 (D) and TeNT H_C (E). Ganglioside complexes where reactivity was not significantly different to that of the component glycolipids are not shown in these graphs.

Fig. 2

(A) Serum A binding to GM1:GD1a is enhanced compared with the individual component gangliosides. There is no significant difference in CTB binding (B) to GM1 compared with any of the GM1 series complexes (“complex independent”). The average signal intensities (n = 3) are quantified (C) with asterisks denoting complexes displaying binding levels significantly different from the sum of the two component glycolipids.

Fig. 3

The mAb MOG1 binds the GM1:GD1a complex on ELISA (A). On PVDF glycoarrays (B), only GD1b containing complexes are bound by the antibody, whilst no signal is seen with the GM1:GD1a complex.

Fig. 4

Siglec-E binds to the neo-epitope formed by GM2:GT1b complex on the PVDF glycoarray, but not to GM2 alone, (A,C) a finding which is also replicated on ELISA (D). Conversely, siglec-F binds to GM2 and to the GM2:GT1b complex on PVDF (B,C), whereas by ELISA reactivity to these target glycolipids is not significantly elevated above background (D). Siglec-7 binding to GD3 is inhibited by GM1 on PVDF-glycoarray, by ELISA and in liposome based assays (E). The average absolute values from each set of three independent experiments have been normalized to 100% to allow comparison on the same histogram. The uncorrected maximum values were 0.34, 1.14 and 70.3% for standard ELISA, liposome ELISA, and PVDF-glycoarray respectively. Error bars represent SEM, corrected for the normalization.