Development of Sialoglycan Recognizing Probes (SGRPs) With Defined Specificities: Towards Exploring the Dynamic Mammalian Sialoglycome

Dense and complex glycans on vertebrate cell surfaces and secreted molecules often terminate with sialic acids (Sias), a diverse family of sugars involved in numerous biological processes, including cell-cell interactions, immune regulation, cellular homeostasis, host-pathogen interactions and microbial pathogeneses. Due to immense structural variations, a major fraction of sialoglycans remain poorly characterized by current inadequate conventional techniques. While traditional glycomic methods are useful, they can destroy or overlook crucial aspects of Sia diversity, or fail to elucidate native structures as they exist in biological systems. To date, in situdetection and analysis of sialoglycans have been largely based on plant lectins, sialidases or antibodies whose preferences are limited and/or uncertain. We defined 9 classes of Sialoglycan Recognizing Probes (SGRPs: SGRP1- SGRP9) aiming to provide a simple and systematic approach to track mammalian sialome changes, using techniques common in most laboratories. Our approach utilizes molecules naturally evolved with maximized specificity towards sialoglycans such as bacterial adhesins, toxin subunits and viral hemagglutinin-esterases. We selected the experimentally proven best candidates for SGRPs and engineered each with a corresponding non-binding mutant as a negative control. Following sialoglycan microarray studies to confirm the binding specificity of our SGRPs, the optimized panel of SGRPs was subsequently applied in common detection methods such as ELISA, Western Blot and FACS analysis. While further work is needed to define a comprehensive suite of SGRPs, our efforts provide a reliable toolkit with specificity to track the mammalian sialoglycome.