Glyco-MAP: a high-throughput multiplexed platform for Glycosylated Mucin Antibody Profiling with glycoengineered cell lines expressing well-defined homogeneous glycostructures - Glyco-MAP: a high-throughput multiplexed platform for Glycosylated Mucin Antibody Profiling with glycoengineered cell lines expressing well-defined homogeneous glycostructures Abstract Early detection is critical to making inroads in the fight against cancer. Tumor antigens elicit tumor associated autoantibodies (TAAb) during early cancer development that can serve as markers for cancer early detection. However, the potential of TAAbs has not been fully realized because previous TAAb studies rely almost exclusively on proteins that lack post-translational modifications, partly due to the lack of appropriate technologies. Mucins are a class of heavily O-glycosylated proteins, whose expression and glycosylation are dysregulated in cancer, making them an excellent target for cancer biomarkers. We propose using mucins as model tumor antigens for the development of a high-through platform assaying TAAbs against glycosylated proteins, namely Glycosylated Mucin Antibody Profiling (Glyco-MAP). The complexity of mucin type O- glycosylation has historically posed challenges in dissecting different forms of endogenous glycosylated mucins. To this end, we will rely on a set of O-glycoengineered cell lines with homogeneous O-glycosylation capacities to express glycoproteins with defined O-glycan structures relevant to cancer. To screen many different glycoproteins in many samples we will use a new method, called Multiplexed In Solution Protein Arrays (MISPA), which combines immunoassays with next-generation sequencing (NGS) to simultaneously quantify hundreds of antibody-antigen interactions. MISPA can work with antigens from any protein expression system if they possess a HaloTag; however, so far it has been used predominately with in vitro expressed unmodified proteins. Glyco- MAP will combine glycoengineered cell lines and MISPA for systematic glycosylated mucin antibody profiling. HaloTagged human mucins will be expressed in O-glycoengineered HEK293 cell lines and covalently “barcoded” with unique DNA sequences. Barcoded mucins and TAAb from patients' sera will be subsequently immunoprecipitated. Individual sample indexing with unique DNA sequences added to protein barcodes, allows for the processing of many samples in parallel. Quantification of DNA barcodes on precipitated antigens and sample indexes using NGS provides a quantitative metric proportional to TAAb concentrations in each sample. Glyco-MAP enables the testing of TAAb abundance against many glycosylated mucins in thousands of patient samples with a single NGS run. We will analyze almost 1 million IgG and IgM antibodies in 400 patients with 2 major epithelial cancers and 400 cancer-free healthy controls against 40 mucins or glycoproteins with a mucin- like domain with 14 distinct glycoforms with cancer relevance. Our proposed study is a close collaboration between experts on proteomics technologies (Dr. Qiu) and glycobiology (Dr. Clausen). This profiling may find new early cancer biomarkers. Understanding the interaction dynamics of glycosylated proteins and how they change in disease is essential for elucidating cellular biology and disease mechanisms. Our study on TAAbs against glycosylated mucins serves as an exemplary application for studying general biological interactions dependent on glycans and unraveling the intricate interactions between the host and microorganisms.
