The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins

Abstract

Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide-and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.O-glycosylation | mucin | protease | glycoproteomics | Siglec

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

“…To assess labeling specificity, we also tested glycoprotein susceptibility towards hydrolytic enzymes. We treated samples with the mucinase StcE that specifically digests highly O-GalNAcylated mucin domains, or with sialidase that removes sialic acid from glycoconjugates (36). Following StcE treatment, the most intense bands labeled by both caged GalNAzMe-1-phosphate 11 and Ac 4 GalNAz feeding had disappeared.…”

“…Supernatant was transferred to a new plate and BCA was used to measure protein concentration. For enzyme treatment, equal amounts of protein (typically 15 µg) were diluted to 40 µL with Lysis Buffer or PBS, treated with either SialEXO (4 µL of a 4 U/µL solution in 50 mM Tris-HCl (pH 6.5), Genovis, Lund, Sweden) or the glycoprotease StcE (50 nM in PBS) (19) and incubated for 2 h at 37 °C. The reaction was quenched by heating to 95 °C for 10 s with subsequent cooling at 4 °C.…”

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

“…Empowered by the proximity labeling approach, we now have generated a priority list of proteins that will be the subject of future work to further reveal the "professional glycoprotein ligands" [14] required for the functional activation of HSCs. Already, preliminary work has shown that basigin plays an active role in hepatic fibrosis [45], and our own work will delve deeper into the molecular nature of the galectin-3-basigin interaction.…”

Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

“…The era of quantitative biology demands great specificity in tools to probe cellular processes or expressed antigens. Biological tools, including antibodies, lectins, engineered binding proteins, or 10 silenced hydrolases have been employed to profile or isolate cellular glycans (45,50). While powerful probes, these proteins suffer from various drawbacks, including the dependence of binding on the structural context or the requirement to simplify glycans before performing binding studies.…”