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.O-glycosylation | mucin | protease | glycoproteomics | Siglec
Conformation-switchable glycopolypeptides were prepared by the living polymerization of glycosylated L-cysteine-N-carboxyanhydride (glyco-C NCA) monomers. These new monomers were prepared in high yield by coupling of alkene-terminated C-linked glycosides of D-galactose or D-glucose to L-cysteine using thiol-ene "click" chemistry, followed by their conversion to the corresponding glyco-C NCAs. The resulting glycopolypeptides were found to be water-soluble and α-helical in solution. Aqueous oxidation of the side-chain thioether linkages in these polymers to sulfone groups resulted in disruption of the α-helical conformations without loss of water solubility. The ability to switch chain conformation and remain water-soluble is unprecedented in synthetic polymers, and allows new capabilities to control presentation of sugar functionality in subtly different contexts.
We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.
We report the development of a new "click"-type reaction for polypeptide modification based on the chemoselective alkylation of thioether groups in methionine residues. The controlled synthesis of methionine polymers and their alkylation by a broad range of functional reagents to yield stable sulfonium derivatives are described. These "methionine click" functionalizations are compatible with deprotection of other functional groups, use an inexpensive, natural amino acid that is readily polymerized and requires no protecting groups, and allow the introduction of a diverse range of functionality and reactive groups onto polypeptides.
We have developed a facile, scalable method for preparation of enzyme responsive copolypeptide vesicles that requires no protecting groups or expensive components. We designed
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.