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All Publications


  • An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proceedings of the National Academy of Sciences of the United States of America Shon, D. J., Malaker, S. A., Pedram, K., Yang, E., Krishnan, V., Dorigo, O., Bertozzi, C. R. 2020

    Abstract

    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. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A 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.

    View details for DOI 10.1073/pnas.2012196117

    View details for PubMedID 32817557

  • Enzyme toolkit for selective enrichment and analysis of mucin-domain glycoproteins Malaker, S. A., Shon, J., Pedram, K., Riley, N. M., Bertozzi, C. R. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2019: S42