An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins D. Judy Shona, Stacy A. Malakera, Kayvon Pedrama, Emily Yanga, Venkatesh Krishnanb, Oliver Dorigob, and Carolyn R. Bertozzia,c,1 aDepartment of Chemistry, Stanford Chemistry, Engineering & Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA 94305; bStanford Women’s Cancer Center, Division of Gynecologic Oncology, Stanford University, Stanford, CA 94305; and cHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305 Edited by James A. Wells, University of California, San Francisco, CA, and approved July 27, 2020 (received for review June 13, 2020) Densely O-glycosylated mucin domains are found in a broad range which mucins and their glycans contribute to disease progression of cell surface and secreted proteins, where they play key physio- remain unclear, in part due to difficulties associated with their logical roles. In addition, alterations in mucin expression and gly- detection and visualization. cosylation are common in a variety of human diseases, such as Existing reagents that are commonly used to stain mucins are cancer, cystic fibrosis, and inflammatory bowel diseases. These agnostic to the unique mucin fold and precise sequence, instead correlations have been challenging to uncover and establish be- depending on charge density or recognition of carbohydrate cause tools that specifically probe mucin domains are lacking. structures. For example, periodic acid-Schiff (PAS) and Alcian Here, we present a panel of bacterial proteases that cleave mucin blue (AB) are standard mucin histochemical stains that react domains via distinct peptide- and glycan-based motifs, generating with monosaccharides. In PAS staining, vicinal diols on sugar a diverse enzymatic toolkit for mucin-selective proteolysis. By mu- residues are oxidized by periodic acid and reacted with Schiff’s tating catalytic residues of two such enzymes, we engineered reagent to yield a distinct magenta color. Alcian blue is a cationic mucin-selective binding agents with retained glycoform prefer- dye that associates with negatively charged sugars and sulfate ences. StcEE447D is a pan-mucin stain derived from enterohemorrha- esters, appearing dark purple when combined with PAS (11). BIOCHEMISTRY gic Escherichia coli that is tolerant to a wide range of glycoforms. Although both reagents are widely used to visualize mucins, their E575A BT4244 derived from Bacteroides thetaiotaomicron is selective mechanisms of action are not intrinsically specific for mucins and for truncated, asialylated core 1 structures commonly associated thus result in significant staining of nonmucin glycoproteins. In with malignant and premalignant tissues. We demonstrated that particular, the PAS technique is known to detect proteoglycans these catalytically inactive point mutants enable robust detection and glycogen, a cytoplasmic constituent of cells that is often and visualization of mucin-domain glycoproteins by flow cytometry, difficult to distinguish morphologically from mucins (12). To Western blot, and immunohistochemistry. Application of our enzy- overcome this issue of specificity, carbohydrate-binding proteins, matic toolkit to ascites fluid and tissue slices from patients with or lectins, are utilized extensively for their ability to bind specific ovarian cancer facilitated characterization of patients based on dif- glycan structures (13). However, because lectins bind to pe- ferences in mucin cleavage and expression patterns. ripherally situated carbohydrates without any additional specificity, O-glycosylation | mucin | mucinase | mass spectrometry | immunohistochemistry Significance Researchers and clinicians rely on robust staining methods to ucin domains are characterized by a high density of gly- detect and visualize biomolecules of interest. Mucins are heavily cosylated serine and threonine residues upon which an M glycosylated proteins expressed on cells throughout the human initiating α-N-acetylgalactosamine (α-GalNAc) is appended and body that have historically proved challenging to stain with high further elaborated. The dense glycosylation twists the protein specificity, hindering attempts to explore their roles in health backbone into a characteristic mucin fold, creating a rigid and and disease. Here, we address this limitation using catalytically extended bottlebrush structure with distinct biophysical properties inactivated mucin-cleaving bacterial proteases as staining re- (1). The clustered glycans themselves form molecular patches that agents. The differing specificities of these enzymes are retained act as surfaces for specific binding interactions (2). Cell surface in their binding properties, enabling a new depth in the analysis and secreted proteins bearing these modular domains extend be- of mucins on cells and patient tissues. yond the canonical 22-member mucin (MUC) family (3). Of the likely hundreds of such proteins, a few specific examples include Author contributions: D.J.S., S.A.M., K.P., and C.R.B. designed research; D.J.S., S.A.M., and PSGL-1, CD43, CD44, CD45, C1-INH, podocalyxin, and Syn- E.Y. performed research; V.K. and O.D. contributed new reagents/analytic tools; D.J.S., CAM1. The mucin domains of these proteins are critical to their S.A.M., K.P., and C.R.B. analyzed data; and D.J.S. and C.R.B. wrote the paper with input functions in embryogenesis (4), host–microbe interplay (5), and from all authors. immune signaling (6). In addition, dysregulation of mucin ex- Competing interest statement: D.J.S., S.A.M., K.P., and C.R.B. are coinventors on a Stan- pression and glycosylation patterns are common in diseases such ford patent application related to this work. C.R.B. is a cofounder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Red- as cancer and inflammatory bowel diseases. Specifically, within the wood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of canonical family of mucins, mucin-1 (MUC1) is overexpressed the Board of Directors of Eli Lilly & Company. O.D. has participated in advisory boards for in >90% of breast tumors (7) and mucin-16 (MUC16)/cancer Tesaro, Merck, and Geneos. O.D. is a speaker for Tesaro and AstraZeneca. antigen 125 (CA125), found at high levels in patient sera, is a key This article is a PNAS Direct Submission. clinical biomarker for treatment efficacy and surveillance in Published under the PNAS license. ovarian cancer (8). In addition to global up-regulation in mucin 1To whom correspondence may be addressed. Email: [email protected]. expression, altered glycan structures are well established as cancer This article contains supporting information online at https://www.pnas.org/lookup/suppl/ antigens, leading to efforts to target these cancer-associated gly- doi:10.1073/pnas.2012196117/-/DCSupplemental. coforms (9, 10). Despite these correlations, the precise contexts in www.pnas.org/cgi/doi/10.1073/pnas.2012196117 PNAS Latest Articles | 1of9 Downloaded by guest on October 4, 2021 lectin-based mucin staining methods require purification of ABSerine proteases mucins from other glycoproteins (e.g., N-glycosylated proteins) Mucinase and glycolipids and fail to distinguish between individual mucin Family S6 proteins. Detection of specific mucins requires the use of anti- Pic from E. coli 042 bodies raised against mucin peptide sequences. Unfortunately, Zinc metalloproteases effective antibodies are not available for all mucin-domain gly- Family M26 coproteins outside of the MUC family, and most of these anti- ZmpC from S. pneumoniae bodies are raised against sequences without glycans (14). Additionally, Family M60 use of antibodies against specific mucins does not provide a BT4244 from B. thetaiotaomicron global picture of mucin composition. Probing the total mucin AM0627 from A. muciniphila AM0908 from A. muciniphila pool of a sample thus requires combinations of these existing tools, AM1514 from A. muciniphila increasing the complexity of sample analysis. As a result of these Family M66 shortcomings, we are currently unable to visualize mucins accu- StcE from E. coli O157:H7 rately, hindering our ability to understand the biological and path- ological roles of mucins. C D We and others have taken advantage of enzymes derived from StcE BT4244AM0627Pic ZmpC AM0908AM1514 StcE BT4244AM0627Pic ZmpC AM0908AM1514 ___ ____ _ __ _ _ _ _ _ _ microorganisms known to colonize niche mucosal environments VC Sia +++++ ++ + VC Sia +++ + + +++ C1-INH Fetuin to develop techniques for selective proteolysis of mucin domains. 97 64 We reported that secreted protease of C1 esterase inhibitor 64 51 (StcE) from enterohemorrhagic Escherichia coli (EHEC) has a 51 39 39 28 distinct peptide- and glycan-based cleavage motif that enables kDa kDa high selectivity for mucins. This mucinase facilitates the digestion of large, densely glycosylated mucin domains into peptides that Fig. 1. Mucin-selective proteases span multiple peptidase families. (A) are more amenable to analysis by mass spectrometry and enables Mucinases enable selective cleavage and removal of mucin-domain glyco- selective release of mucin fragments from biological samples proteins from biological samples with distinct peptide- and glycan-dependent (15). Based on the rich history of binding reagents derived from activities. (B) Candidate mucinases from pathogenic and commensal bac- inactivated hydrolases (16–18), we reasoned that catalytically teria