Cell-based mucin arrays

The cell-based mucin array is a versatile platform for expressing and interrogating recombinant mucin reporter proteins with representative patterning and customizable O-glycan structures. The platform is based on glycoengineered mammalian cell lines (HEK293/CHO), in which the glycosylation machinery is genetically rewritten to enable controlled display of specific O-glycan core structures and terminal epitopes with defined sialylation, fucosylation, and sulfation. Uniquely, the platform presents glycans in their native protein context, enabling investigation of how O-glycan density, clustering, and multivalency influence interactions with mucins. A conceptual framework for recognition of such glycan-context cues is provided by the patterned arrangement of O-glycans within mucin O-glycodomains, often composed of tandem repeat (TR) sequences with distinct O-glycosites resembling molecular barcodes. Mucin reporters mimic the serine-, threonine-, and proline-rich domains of natural mucins and mucin-like proteins or they can be designed as artificial Glycocarriers with model O-glycan cluster motifs. Reporters expressed as membrane-bound forms for cell display or as secreted fusion proteins for production can be applied in diverse bioassays. They have been used to probe glycan/mucin binding by viral and microbial adhesins, as well as human Siglec immune receptors. Moreover, the platform provides defined substrates for functional analyses of mucin and O-glycodomain degradation by microbial O-glycopeptidases or mucinases, revealing information of substrate specificities, cleavage points, and catalytic mechanisms. This chapter describes how the cell-based mucin array can be used to dissect interactions with mucins by glycan- and mucin-binding receptors as well as mucinases. The platform overcomes limitations of contemporary technologies by enabling studies with mimics of natural mucins and O-glycodomains that preserve clustered and patterned O-glycan contexts.