Abstract
Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.
Highlights
Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids
The entire sequences selected as representative for each of the human mucin tandem repeat (TR) O-glycodomains are shown in Supplementary Fig. 1 and Table 1, which illustrates that mucin TRs are imperfect in sequence but present characteristic patterns of O-glycans
We found evidence that these adhesins showed binding selectivity for mucin TRs expressed in HEK293WT, and to explore these findings further we here included binding studies with the preferred O-glycan structures on different mucin TRs (Fig. 4a)
Summary
Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are a large family of heavily glycosylated proteins that line all mucosal surfaces and represent the major macromolecules in body fluids[2]. State-of-the-art technologies to capture the informational content of mucins are confined to studies with synthetic and isolated O-glycans[10], synthetic and chemoenzymatically produced short glycopeptides[11], and synthetic glycopeptides as well as non-natural polymers[12,13]; all of which are rare commodities that do not reflect the complex information captured in distinct human mucins by their display of patterns and structures of O-. With the advent of the facile nuclease-based gene engineering technologies, it has become possible to engineer mammalian cells with combinatorial knockout (KO) and knockin (KI)
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