Microarray analyses of closely related glycoforms reveal different accessibilities of glycan determinants on N-glycan branches

Abstract

Glycans mediate a wide variety of biological roles via recognition by glycan-binding proteins (GBPs). Comprehensive knowledge of such interaction is thus fundamental to glycobiology. While the primary binding feature of GBPs can be easily uncovered by using a simple glycan microarray harboring limited numbers of glycan motifs, their fine specificities are harder to interpret. In this study, we prepared 98 closely related N-glycoforms that contain 5 common glycan epitopes which allowed the determination of the fine binding specificities of several plant lectins and anti-glycan antibodies. These N-glycoforms differ from each other at the monosaccharide level and were presented in an identical format to ensure comparability. With the analysis platform we used, it was found that most tested GBPs have preferences toward only one branch of the complex N-glycans, and their binding toward the epitope-presenting branch can be significantly affected by structures on the other branch. Fine specificities described here are valuable for a comprehensive understanding and applications of GBPs.

Terminal Epitope-Dependent Branch Preference of Siglecs Toward N-Glycans

“…Unless otherwise stated, all chemicals were purchased and used without further purification. The 98 N -glycan microarray was prepared as described previously ( Supplementary Figure S1 ) ( Li et al, 2019 ). Sugar nucleotides, including uridine 5′-diphospho-galactose (UDP-Gal) ( Muthana et al, 2012 ), were prepared as described previously.…”

“…Product-containing fractions were pooled and lyophilized. The quantifications of AEAB-labeled glycans were conducted as previously described ( Li et al, 2019 ).…”

“…The 98 N -glycan microarray slide ( Li et al, 2019 ) was fitted with a ProPlate 8-well microarray module (Sigma-Aldrich), and the subarrays were then rehydrated for 10 min with 200 μL of Buffer TSMTB (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.05% (v/v) Tween-20, and 1% (w/v) BSA) at room temperature. Then, the buffer was drained and 200 μL of Siglec-3, -8, -9, -10, and -F (R&D Systems) (20 µg/ml) in TSMTB were added into each subarray, sealed, and incubated at room temperature for 1 h with gentle shaking.…”

Recent Advances in the Chemical Biology of N-Glycans

“…Meanwhile, recent advances in the preparation of the whole structure of various N -glycans have allowed the full realization of structure–activity relationships, elucidating the significance of complexity of N -glycan structures ( Figure 4 ). For example, these advances have provided insights into the differences in the lectin recognition of each branch [ 49 , 50 , 51 , 52 , 53 , 54 ], the improvement of affinity due to the inclusion of multiple recognition units (multivalent effect) [ 20 , 21 , 55 , 56 , 57 , 58 , 59 ], the influence of chain length on affinity [ 60 , 61 ], and remote (heterovalent) recognition [ 59 ].…”

“…Wang et al demonstrated the differences of each N -glycan branch in lectin recognition by comprehensive interaction analysis of various N -glycans with several lectins using a glycan array [ 53 , 54 ]. Plant-derived Sambucus nigra lectin (SNA), which recognizes sialic acid, recognized the sialic acid on the α1,3-branched chain more strongly than the sialic acid on the α1,6-branched chain.…”