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广东快乐十分今天开奖:The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins
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Mucin-domain glycoproteins are found in nearly every tissue of the human body, and are important in biological processes ranging from embryogenesis to cancer. Because there are few tools to study mucin domains, their biological functions at the molecular scale remain unclear. Here, we help address a hurdle to the study of mucin-domain glycoproteins by characterizing a bacterial protease with selectivity for mucins. This mucinase selectively removes native mucins from cell surfaces and cuts them into fragments amenable to analysis.
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.
?1S.A.M. and K.P. contributed equally to this work.
- ?2To whom correspondence should be addressed. Email: .
Author contributions: S.A.M., K.P., and C.R.B. designed research; S.A.M., K.P., M.J.F., and B.A.B. performed research; V.K., C.P., J.Y., J.R.K., U.W., and O.D. contributed new reagents/analytic tools; S.A.M., K.P., M.J.F., B.A.B., E.C.W., and C.R.B. analyzed data; and S.A.M., K.P., and C.R.B. wrote the paper.
Conflict of interest statement: A patent application relating to the use of enzymes to digest mucin-domain glycoproteins has been filed by Stanford University (docket no. STAN-1510PRV). C.R.B. is a cofounder and Scientific Advisory Board member of Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly & Company. O.D. has participated in advisory boards for Tesaro, Merck, and Geneos. O.D. is a speaker for Tesaro and AstraZeneca.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1813020116/-/DCSupplemental.
Published under the PNAS license.