Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 15, Pages 7278-7287Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1813020116
Keywords
O-glycosylation; mucin; protease; glycoproteomics; Siglec
Categories
Funding
- National Cancer Institute [R01CA227942]
- National Institute of General Medical Sciences F32 Postdoctoral Fellowships
- National Science Foundation Graduate Research Fellowship
- Stanford Graduate Fellowship
- Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program
- National Institutes of Health [IA106987, CA221244]
- American Heart Association [17SDG33660424]
- Mary Lake Polan Gynecologic Oncology Endowment for Gynecologic Cancer Research, Stanford School of Medicine, Stanford University
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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 Cl 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.
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