Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 35, Pages 21299-21307Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2012196117
Keywords
O-glycosylation; mucin; mucinase; mass spectrometry; immunohistochemistry
Categories
Funding
- National Cancer Institute Grant [R01CA200423]
- National Science Foundation Graduate Research Fellowship
- National Science Foundation Stanford Graduate Fellowship
- National Institute of General Medical Sciences F32 Postdoctoral Fellowship [F32GM126663-01]
- Stanford Chemistry, Engineering & Medicine for Human Health (ChEM-H) Chemistry/Biology Interface Predoctoral Training Program
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Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StCE(E447D) is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244(E575A) derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.
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