MucinsMucins constitute a large family of heavily glycosylated proteins that line all mucosal surfaces and form crosslinked networks or linear bundles that constitute the major macromolecules in body fluids1). Mucins clear, contain, feed, direct, and continuously replenish our microbiomes, limiting unwanted co-habitation and repressing harmful pathogenic microorganisms2). Mucin O-glycans present the essential binding opportunities and informational cues for microorganisms via their adhesins, however, our understanding of these features is essentially limited to results from studies with simple oligosaccharides without the protein context of mucins and the higher order features presented by dense O-glycan motifs. Mucins are notoriously difficult to isolate due to their size and heterogeneity, and production by recombinant expression in cell lines is impeded due to difficulties with the assembly of full coding expression constructs often resulting in heterogeneous products. There are over 20 distinct human genes encoding membrane bound and secreted mucins in addition to many genes encoding glycoproteins with mucin-like domains. The large secreted (gel-forming) mucins form oligomeric networks or extended bundles through inter-and intramolecular disulfide bridges in the C- and N-terminal cysteine-rich regions1). A common characteristic of all mucins is that a major part of the extracellular region is comprised of variable TRs that carry dense O-glycans. The main binding cues for lectins and adhesins of the microbiota lie in these TR regions that position dense arrays of O-glycans in unique patterns. Interestingly, the TR regions appear poorly conserved throughout evolution in contrast to the flanking regions of the large mucins, which has been interpreted to reflect that the TR regions simply need to carry dense O-glycans without specific patterns. An alternative interpretation is that divergence in TR sequences has co-evolved with the microbiota to govern refined interactions with larger motifs of O-glycan patterns as recently suggested for streptococcal serine-rich adhesins3). The TR regions of mucins are quite distinct in length and in sequences with distinct spacing of O-glycosites, and TRs in any mucin exhibit individual variability in numbers as well as to some degree in actual sequences. We hypothesized that mucin TRs can present higher order glycan-binding motifs – clustered saccharide patches or discontinuous glycan epitopes – encoded by the combination of the distribution of O-glycans (patterns) and specific O-glycans (structures). Thus, we envisioned opportunities for unique “codes” in mucin TRs, governed by particular display of patterns and structures of O-glycans, which could provide a much greater potential binding epitome or catalog of cues than the comparatively limited repertoire of binding epitopes comprised of simple oligosaccharide motifs available in humans. The facile nuclease-based gene engineering technologies (CRISPR/Cas9 and ZFN) has made it possible to perform comprehensive custom engineering of glycosylation capacities in mammalian cells. A unique glycoengineered isogenic cell libraries with combinatorial knockout (KO) and knockin (KI) of glycosylation genes were previously developed to display subsets and distinct features of the glycome on the cell surface or on secreted recombinant proteins to probe biological interactions dependent on glycans4). Importantly, such cell-based display strategies allow for presentation of glycans in the The cell-based mucin display platform – Mucin Display –Mucins arguably represent the last frontier in the analytics of glycoproteins. Most mucins are extremely large and heterogeneous glycoproteins that are resistant to conventional glycoproteomics strategies dependent on proteolytic fragmentation and sequencing. Currently, there are no methods for obtaining human mucin molecules in reasonable purity, with defined glycans, and this is a fundamental barrier and limitation for studies of mucins and their complex biology as well as for the microbiome field. We therefore sought to capture the molecular cues contained in human mucin and mucin-like O-glycodomains (TRs) and enable molecular dissection of these cues by developing a cell-based platform for the display and production of representative mucin TRs with defined O-glycans to molecularly dissect the roles in microbial interaction with the mucin sequence and glycosylation independently. 112Display of the Human Mucinome with Defined O-Glycans by Gene Engineered Cells– Potential for mucin therapeuticsYoshiki NarimatsuCopenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen, Denmark
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