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<p>Mucins are a class of highly O-glycosylated proteins that are ubiquitously expressed on cellular
surfaces and are important for human health, especially in the context of carcinomas. However,
the molecular mechanisms by which aberrant mucin structures lead to tumor progression and
immune evasion have been slow to come to light, in part because methods for selective mucin
degradation are lacking. Here we employ high resolution mass spectrometry, polymer synthesis,
and computational peptide docking to demonstrate that a bacterial protease, called StcE,
cleaves mucin domains by recognizing a discrete peptide-, glycan-, and secondary structure-
based motif. We exploited StcE’s unique properties to map glycosylation sites and structures of
purified and recombinant human mucins by mass spectrometry. As well, we found that StcE will
digest cancer-associated mucins from cultured cells and from ovarian cancer patient-derived
ascites fluid. Finally, using StcE we discovered that Siglec-7, a glyco-immune checkpoint
receptor, specifically binds sialomucins as biological ligands, whereas the related Siglec-9
receptor does not. Mucin-specific proteolysis, as exemplified by StcE, is therefore a powerful
tool for the study of glycoprotein structure and function and for deorphanizing mucin-binding
receptors.
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A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry