Development and Commercialization of Sialoglycan-Recognizing Probes

Project Summary/Abstract Sialoglycans or Sias, which are abundant on cell surfaces and in secreted molecules, play key roles in various biological processes, interact with both commensals and pathogens, and change over time in response to environmental signals. However, most of this complexity and variation remains poorly characterized by traditional techniques, using plant lectins, sialidases or antibodies, whose preferences are limited and/or uncertain. Meanwhile mass spectrometry methods can overlook crucial aspects of Sia diversity and/or fail to elucidate native Sia structures in biological systems. To address these challenges, the U01CA199792 grant supported by the NIH Common Fund Glycoscience Program took advantage of naturally evolved microbial molecules (bacterial adhesins, toxin subunits and viral hemagglutinin-esterases) that can recognize sialoglycans with defined specificity. Multiple classes of Sialoglycan Recognizing Probes (SGRPs) with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding non-binding mutant as a negative control. The optimized panel of SGRPs can be used in methods common in most bioscience labs, such as ELISA, western blot, flow cytometry and histochemistry. In Phase I, we propose continued development and validation of six SGRPs (SGRP1, SGRP2, SGRP3, SGRP5, SGRP6, and SGRP8). We will reproduce SGRP1, SGRP2, SGRP3, and SGRP8 based on previously established methods and further validate and optimize their applications in various laboratory techniques across diverse biological sample types. SNA, a plant-derived lectin, was selected as SGRP6 due to its unmatched specificity for α2-6 sialoglycans. Since no other microbial molecules surpass its specificity, we will develop a recombinant SNA protein with an AviTag to overcome the limitations of the plant-derived form, ensuring consistent and reproducible production. We will also develop a non-binding mutant control for the recombinant SNA. SGRP5 is an affinity-purified chicken IgY polyclonal antibody against Neu5Gc sialoglycans, which is limited for long-term use due to finite supply and risk of future non-reproducibility. We propose to develop chicken IgY monoclonal antibodies targeting all common forms of Neu5Gc sialoglycans, providing a more durable and reproducible solution for routine use. In Phase II, we will further develop and optimize SGRPs (SGRP4, SGRP7, and SGRP9) that specifically recognize 4, 7, and 9-O-acetylated sialoglycans, which are common modifications present in many mammalian cells. SGRP probes could revolutionize the analysis of sialoglycans by making it simpler, more efficient, affordable and accessible, allowing nonexperts using common laboratory techniques to find interesting sialome patterns in biological systems. They also provide tools for real-time monitoring of glycosylation patterns in biopharmaceutical production, ensuring consistency and quality. Our goal is to ensure these probes become readily available to the research community, driving progress across multiple biomedical disciplines.