@article {66017, title = {An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins}, journal = {Proceedings of the National Academy of Sciences}, volume = {117}, year = {2020}, month = {Jan-09-2020}, pages = {21299 - 21307}, 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.

}, issn = {0027-8424}, doi = {10.1073/pnas.2012196117}, url = {http://www.pnas.org/lookup/doi/10.1073/pnas.2012196117https://syndication.highwire.org/content/doi/10.1073/pnas.2012196117http://www.pnas.org/syndication/doi/10.1073/pnas.2012196117}, author = {Shon, D. Judy and Malaker, Stacy A. and Pedram, Kayvon and Yang, Emily and Krishnan, Venkatesh and Dorigo, Oliver and Bertozzi, Carolyn R.} } @article {66012, title = {The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins}, journal = {Proceedings of the National Academy of Sciences}, volume = {116}, year = {2019}, month = {Sep-04-2019}, pages = {7278 - 7287}, 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{\textquoteright}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.

}, issn = {0027-8424}, doi = {10.1073/pnas.1813020116}, url = {http://www.pnas.org/lookup/doi/10.1073/pnas.1813020116https://syndication.highwire.org/content/doi/10.1073/pnas.1813020116http://www.pnas.org/syndication/doi/10.1073/pnas.1813020116}, author = {Malaker, Stacy A. and Pedram, Kayvon and Ferracane, Michael J. and Bensing, Barbara A. and Krishnan, Venkatesh and Pett, Christian and Yu, Jin and Woods, Elliot C. and Kramer, Jessica R. and Westerlind, Ulrika and Dorigo, Oliver and Bertozzi, Carolyn R.} }