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The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.
Proc Natl Acad Sci U S A 2019; 116(15):7278-7287PN

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'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.

Authors+Show Affiliations

Department of Chemistry, Stanford University, Stanford, CA 94305.Department of Chemistry, Stanford University, Stanford, CA 94305.Department of Chemistry, University of Redlands, Redlands, CA 92373.Department of Medicine, San Francisco Veterans Affairs Medical Center and University of California, San Francisco, CA 94143.Stanford Women's Cancer Center, Division of Gynecologic Oncology, Stanford University, Stanford, CA 94305.Leibniz-Institut für Analytische Wissenschaften (ISAS), 44227 Dortmund, Germany. Department of Chemistry, Umeå University, 901 87 Umeå, Sweden.Leibniz-Institut für Analytische Wissenschaften (ISAS), 44227 Dortmund, Germany.Department of Chemistry, Stanford University, Stanford, CA 94305.Department of Bioengineering, University of Utah, Salt Lake City, UT 84112.Leibniz-Institut für Analytische Wissenschaften (ISAS), 44227 Dortmund, Germany. Department of Chemistry, Umeå University, 901 87 Umeå, Sweden.Stanford Women's Cancer Center, Division of Gynecologic Oncology, Stanford University, Stanford, CA 94305.Department of Chemistry, Stanford University, Stanford, CA 94305; bertozzi@stanford.edu. Howard Hughes Medical Institute, Stanford, CA 94305.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

30910957

Citation

Malaker, Stacy A., et al. "The Mucin-selective Protease StcE Enables Molecular and Functional Analysis of Human Cancer-associated Mucins." Proceedings of the National Academy of Sciences of the United States of America, vol. 116, no. 15, 2019, pp. 7278-7287.
Malaker SA, Pedram K, Ferracane MJ, et al. The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. Proc Natl Acad Sci USA. 2019;116(15):7278-7287.
Malaker, S. A., Pedram, K., Ferracane, M. J., Bensing, B. A., Krishnan, V., Pett, C., ... Bertozzi, C. R. (2019). The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. Proceedings of the National Academy of Sciences of the United States of America, 116(15), pp. 7278-7287. doi:10.1073/pnas.1813020116.
Malaker SA, et al. The Mucin-selective Protease StcE Enables Molecular and Functional Analysis of Human Cancer-associated Mucins. Proc Natl Acad Sci USA. 2019 04 9;116(15):7278-7287. PubMed PMID: 30910957.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. AU - Malaker,Stacy A, AU - Pedram,Kayvon, AU - Ferracane,Michael J, AU - Bensing,Barbara A, AU - Krishnan,Venkatesh, AU - Pett,Christian, AU - Yu,Jin, AU - Woods,Elliot C, AU - Kramer,Jessica R, AU - Westerlind,Ulrika, AU - Dorigo,Oliver, AU - Bertozzi,Carolyn R, Y1 - 2019/03/25/ PY - 2019/09/25/pmc-release PY - 2019/3/27/pubmed PY - 2019/5/23/medline PY - 2019/3/27/entrez KW - O-glycosylation KW - Siglec KW - glycoproteomics KW - mucin KW - protease SP - 7278 EP - 7287 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 15 N2 - 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. SN - 1091-6490 UR - https://www.unboundmedicine.com/medline/citation/30910957/The_mucin-selective_protease_StcE_enables_molecular_and_functional_analysis_of_human_cancer-associated_mucins L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=30910957 DB - PRIME DP - Unbound Medicine ER -