A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

• Published in: Molecules (Basel, Switzerland) Switzerland), 2021-10, Vol.26 (20), p.6194
• Main Authors: Zhang, Libo, Yu, Hai, Bai, Yuanyuan, Mishra, Bijoyananda, Yang, Xiaoxiao, Wang, Jing, Yu, Evan B, Li, Riyao, Chen, Xi
• Format: Article
• Language: English
• Subjects: Antigens; Arrays; Bacterial diseases; Binding; Blood Proteins - chemistry; Blood Proteins - metabolism; Bovine serum albumin; Carbohydrate Conformation; Carbohydrate Sequence; carbohydrate-protein conjugate; Carbohydrates; Conjugates; Enzymes; Fluorescence; Fluorescent Dyes; galectin; Galectin-3; Galectins - chemistry; Galectins - metabolism; Glucose; Glycan; glycan-binding protein; Glycated Serum Albumin; Glycation End Products, Advanced; Glycoproteins; Humans; Immobilization; Immobilized Proteins; lectin; Lectins; Magnetic Phenomena; Magnetic properties; Mammals; multiplex assay; Multiplexing; Plant Lectins - chemistry; Plant Lectins - metabolism; Polysaccharides - chemistry; Protein Array Analysis - methods; Protein Binding; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serum Albumin; Serum Albumin, Bovine; Streptococcus infections; Vaccines
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules26206194

Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.