Carbohydrate Chips for Studying High-Throughput Carbohydrate−Protein Interactions

Abstract

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.

Printed covalent glycan array for ligand profiling of diverse glycan binding proteins

“…By comparing galectin-4 binding to saturated glycans (printed at 100 M concentration) with binding to subsaturated glycans (printed at 10 M concentration), preferred binding specificities were revealed. In particular, Gal␣1-3-linked to lactose (35-37), Fuc␣1-2-linked to lac(NAc) (100, 103, and 105-107), or R-GlcNAc␤1-3-linked to lactose (123), as well as 3Ј sulfation (11)(12)(13)(14)(15)(16), substantially enhanced the affinity. This specificity profile is similar to that reported for a rat ortholog of galectin-4 (48, 49).…”

“…Each format differs in the type of glycans and the manner in which they are displayed. Some use noncovalent attachment to plastic or nitrocellulose membrane (6)(7)(8)(9)(10)(11)(12), and others use covalent attachment to plastic, gold, or glass (6,(13)(14)(15)(16)(17)(18)(19)(20). The glycans displayed range from a limited number of 5-45 exemplary structures representing terminal sequences on glycoprotein or glycolipid glycans (6,7,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), to libraries of defined glycans, proteoglycan fragments, and microbial polysaccharides (8)(9)(10).…”

Design of Triazole‐Tethered Glycoclusters Exhibiting Three Different Spatial Arrangements and Comparative Study of their Affinities towards PA‐IL and RCA 120 by Using a DNA‐Based Glycoarray

“…In order to provide a quantitative analysis of the binding affinities between lectin RCA 120 and glycoconjugates 22 and 23, the corresponding IC 50 values were measured as previously reported [41] and compared with the IC 50 values determined for 24 a and 24 b. [13] We determined IC 50 values as the concentration of lactose required for the removal of 50 % of RCA 120 bound to the immobilized glycoconjugates.…”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…Microarray coatings of type 2B can be prepared using a variety of chemically different linker systems including glutaraldehyde ("aldehyde slides"), phenylendiisothiocyanate, PEGs of different molar mass, or various heterobifunctional crosslinker [7,22,[25][26][27][28][29][30][31]. To date, only a few of these slide surfaces are commercially available.…”