An Enzymatic Toolkit for Selective Proteolysis, Detection, and Visualization of Mucin-Domain Glycoproteins
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 spanning four MEROPS peptidase families were selected for charac- terization. (C) IRDye 800CW-labeled C1-INH and (D) IRDye 800CW-labeled inactivated mucinases could be leveraged as mucin-selective fetuin were treated with mucinases at a 1:50 E:S ratio ± 10 nM VC Sia for staining reagents. Ideally, these reagents would retain the pep- 16 h at 37 °C, and digests were separated by SDS/PAGE. Cleavage prod- tide and glycoform specificities of their parent enzymes, pro- ucts were visualized using an Odyssey CLx Near-Infrared Fluorescence viding the research community with a panel of stains. Imaging System. We found that mucinases derived from both pathogenic and commensal microorganisms exhibited unique peptide- and A glycan-based cleavage motifs (Fig. 1 ). Catalytic point mutants against the mucin-domain–containing glycoprotein C1 esterase of select mucinases retain binding specificity and can be used as inhibitor (C1-INH). A majority of the candidates were able to staining reagents for flow cytometry, Western blot, and immuno- cleave C1-INH at an enzyme-to-substrate (E:S) ratio of 1:50. A histochemistry, enabling the elucidation of dynamic changes in subset of these proteases, specifically AM0627 and BT4244, mucin expression in a glycosylation-dependent manner. These demonstrated an increase in activity after Vibrio cholerae sialidase engineered mucinases represent mucin-selective staining re- (VC Sia) treatment of C1-INH (Fig. 1C), suggesting their sen- agents for the visualization of global mucin composition. Suc- sitivity to the presence of sialic acid. Raising the E:S ratio to 1:1 cessful application of our enzyme-based mucin staining strategy enabled cleavage by AM0908, AM1514, and Pic and significantly to primary tissue from patients highlights the potential for this improved cleavage of desialylated C1-INH by BT4244 (SI Ap- approach to deepen our understanding of basic mucin biology pendix, Fig. S2). To probe the enzymes’ selectivity for mucin- and bulk changes that result in disease states. domain glycoproteins, we tested their ability to cleave fetuin, Results which is O-glycosylated, but is not a mucin (32). At an E:S ratio of 1:50, all proteases, with the exception of ZmpC, showed no Bacterial Mucinases Exhibit Unique Peptide- and Glycan-Based Cleavage D Motifs. To maximize the likelihood of identifying useful stains, and activity against fetuin (Fig. 1 ), suggesting that they are, in fact, to explore the diversity of cleavage specificities exhibited by mucin- mucin selective. A concentration screen with ZmpC demon- degrading bacteria, we began by expanding the existing mucinase strated selectivity for C1-INH over fetuin within a specific E:S SI Appendix toolkit. Families of mucin-targeting proteases that have been ratio range ( , Fig. S3), indicating that ZmpC is also reported in the microbiology literature include the S6 serine pro- selective for mucins, but at a much lower concentration. Im- tease autotransporters of Enterobacteriaceae (SPATEs) and M26, portantly, the resulting cleavage patterns were unique between M60-like, and M66 zinc metalloproteases (19–22). We chose different mucin-selective proteases, including some sensitivity to BT4244 from Bacteroides thetaiotaomicron (21), protein involved in sialic acid, indicating potential peptide and/or glycan specificities intestinal colonization (Pic) from enteroaggregative E. coli 042 (23), distinct from that of StcE (15). and zinc metalloproteinase C (ZmpC) from Streptococcus pneu- In a similar manner to how we previously characterized StcE, moniae (24), which have all been shown to exhibit activity against we investigated the enzymes’ specific cleavage motifs using mass mucins. In addition to these known proteases, we chose to study spectrometry (MS). The mucinases were reacted with the uncharacterized M60-like proteases AM0627, AM0908, and AM1514 recombinant mucin-domain glycoproteins leukosialin (CD43), from Akkermansia muciniphila, a gut symbiont found within the hu- P-selectin glycoprotein ligand 1 (PSGL-1), MUC16, and podo- man and mouse gut mucus layer that has been shown to rely pref- calyxin. As BT4244 and AM0627 demonstrated a sensitivity to erentially on host-derived mucinasanenergysource(25)andhas sialic acid, we also digested the mucin-domain glycoproteins in been linked to inflammatory bowel diseases (26, 27), obesity (28, 29), the presence of a sialidase. Following mucinase digestion (with autism (30), and cancer (31). and without sialidase), the peptides were de-N-glycosylated with We expressed and purified these recombinant bacterial pro- PNGaseF, trypsinized, and subjected to MS analysis (Fig. 2A). teases (SI Appendix, Fig. S1) spanning four distinct MEROPS By manually comparing peptides present in the mucinase-treated peptidase families (Fig. 1B). We then tested each protease samples but not the control (PNGaseF and trypsin-only)
2of9 | www.pnas.org/cgi/doi/10.1073/pnas.2012196117 Shon et al. Downloaded by guest on October 4, 2021 samples, we found that each mucinase has a distinct peptide- and biological samples and specific digestion of mucin-domain gly- glycan-based cleavage motif (Fig. 2B–H and Datasets S1–S6). coproteins for mass spectrometry. BT4244 cleaved peptides N-terminally to glycosylated Ser or Thr residues and preferred the modified residue to bear a Catalytically Inactive Mucinases Detect Live Cell Surface Mucin truncated, tumor-associated glycan such as the Tn (O-GalNAc) Populations by Flow Cytometry. To generate mucin-selective or T (O-GalNAc-galactose) antigen. Interestingly, AM0627 binding agents, we first mutated the catalytic glutamate resi- cleaved between two O-glycosylated Ser or Thr residues bearing dues of StcE (E447D) (15), BT4244 (E575A) (22), and AM0627 SI Appendix similar structures. As expected, these enzymes often did not (E326A) and confirmed loss of catalytic activity ( , cleave when the Ser or Thr residue was modified by sialic acid, Fig. S5). StcE was selected as our pan-mucin stain based on its although we observed a few examples of this phenomenon (in- ease of expression, high activity, stability, and vast range of tol- dicated by brackets around the sialyl-T structure; Fig. 2C and D). erated glycoforms, which has previously been justified through Pic had a similar cleavage pattern to BT4244; however, this structural biology and molecular modeling (15, 33). BT4244 and enzyme showed absolutely no tolerance for the presence of sialic AM0627 were chosen for their unique recognition of truncated and desialylated mucin-type O-glycopeptides. The strong pref- acid (Fig. 2E). Conversely, ZmpC was agnostic to the presence of erence of BT4244 for the Tn antigen is corroborated by a pre- sialic acid and was unique in that it cleaved the peptide backbone viously published crystal structure of the catalytic domain (22) several amino acids away from the glycosylated residue (Fig. 2F). showing a binding pocket that accommodates an α-GalNAc Finally, two additional enzymes from A. muciniphila, AM0908 residue whereas binding to the sialyl-Tn (STn) antigen is steri- and AM1514, cleaved N-terminally to a glycosylated Ser or Thr, cally blocked. The remaining mucinases were not pursued due to with a marked preference for an adjacent Ser or Thr residue. G either redundancy in activity or lower purification yields. While AM0908 tolerated sialic acid (Fig. 2 ), AM1514 was To test the binding ability of each point mutant, we used H specific for T and Tn antigen-modified amino acids (Fig. 2 ). chemically conjugated Alexa Fluor 647 (AF647) constructs and I Fig. 2 depicts a CD43 peptide with the mucinase cleavage sites checked for cell surface staining via live cell flow cytometry. SI Appendix mapped onto it, and , Fig. S4 demonstrates full We first employed GALE knockout (KO) K562 cells to test AF647- cleavage maps of all four mucin-domain glycoproteins. Excel StcEE447D binding, as these cells are deficient in UDP-glucose/ files listing all cleaved peptides can be found in Datasets S1–S6. galactose-4-epimerase (GALE) and are thus unable to inter- Although we used recombinant mucin-domain glycoproteins, convert UDP-glucose and UDP-GlcNAc to UDP-galactose and which have simpler glycans than endogenous mucins, these UDP-GalNAc, respectively. These cells are still able to use mucinase-MS studies were informative because many of these media-derived glucose to synthesize nucleotide sugars of glucose, BIOCHEMISTRY proteins are known to be glycosylated, but very little is known mannose, sialic acid, and fucose, but are incapable of initiating or about site specificity or glycan structure. Together, these en- extending glycans with GalNAc or galactose (Gal). This pheno- zymes represent a mucinase toolkit with diverse specificities and, type can, however, be rescued with media supplementation of as such, will find utility in selective depletion of mucins from 10 μM galactose or 100 μM GalNAc (34). AF647-StcEE447D
A B StcE C BT4244
Manual () Glycopeptide Motif MS Sequencing Generator Mucinases
DEAM0627 Pic FZmpC GAM0908
()
H AM1514 I + Sialidase
M S S V P Q E T P H A T S H P A V P I T A N S L G S H T V T G G T I T T N S P E T S S R - Sialidase
M S S V P Q E T P H A T S H P A V P I T A N S L G S H T V T G G T I T T N S P E T S S R
Fig. 2. Mucinase consensus motifs determined by mass spectrometry. (A) Workflow for generating consensus sequences. Four recombinant mucin-domain glycoproteins were digested with mucinases, de-N-glycosylated, trypsinized, and subjected to MS analysis. Peptides present in the mucinase-treated samples were used as input for weblogo.berkeley.edu (±5 residues from the site of cleavage). (B–H) Minimum cleavage motifs for mucinases. (B) StcE is a pan-mucinase that cleaves in a T/S*-X-T/S pattern, where the asterisk denotes a glycosylation site. Adapted from ref. 15. (C) BT4244’s consensus motif, representative of 192 unique cleavage sites. (D) AM0627’s consensus motif, representative of 60 unique cleavage sites. (E)Pic’s consensus motif, representative of 38 unique cleavage sites. (F) ZmpC’s consensus motif, representative of 74 unique cleavage sites. (G) AM0908’s consensus motif, representative of 95 unique cleavage sites. (H) AM1514’s consensus motif, representative of 69 unique cleavage sites. (I) All of the mucinase cleavage sites mapped onto a peptide from CD43, with and without sialidase treatment. Sites of glycosylation are annotated with a yellow square (GalNAc). Larger structures are not indicated but were present in some instances.
Shon et al. PNAS Latest Articles | 3of9 Downloaded by guest on October 4, 2021 showed weak binding to unrescued or galactose-rescued GALE AF647-StcEE447D 1.5 KO K562s. Supplementation with GalNAc led to a 3.1-fold in- A )
5 GALE KO crease in mean fluorescence intensity (MFI), emphasizing the 1 requirement for GalNAc-initiated mucin-type O-glycosylation. GALE KO+Gal GALE KO+GalNAc Full rescue with both galactose and GalNAc, which facilitates 0.5 complete elaboration of glycans, resulted in a 12.8-fold increase MFI (x10 GALE KO+Gal+GalNAc E447D 0 102 104 106 A 0 in binding (Fig. 3 ). Further, to test whether StcE could be Fluorescence (AU) used to probe changes in cell surface mucin levels in response to perturbations, we stained wild-type (WT) K562s pretreated with B AF647-BT4244E575A 10
mucinases and saw decreased signal when mucins were removed ) 8 5 WT enzymatically or blocked using catalytic point mutant mucinases 6 WT+VC Sia (SI Appendix, Fig. S6). 4 COSMC KO
Based on the cleavage motifs for BT4244 and AM0627, we chose MFI (x10 2 COSMC KO+VC Sia to test binding on WT and COSMC KO K562 cells. COSMC 2 4 6 0 10 10 10 0 (C1GALT1C1) is the unique chaperone of core 1 T-synthase, or Fluorescence (AU) core 1 β3-galactosyltransferase (β3GalT), which is responsible for transferring galactose to GalNAc-α-Ser/Thr to form the T antigen. C E Knockout of COSMC leads to aggregation and proteasomal deg- PBS StcE PBS StcE PBS StcE PBS StcE radation of nascent T-synthase, preventing extension of glycans be- BSAfetuinC1-INHpodocalyxinMUC16PSGL-1CD45 BSAfetuinC1-INHpodocalyxinMUC16PSGL-1CD45 yond the Tn and STn antigens (35). AF647-BT4244E575A binding to 260 260 wild-type K562s increased 14.2-fold with VC Sia treatment, high- 125 lighting the preference of the protease for desialylated substrates. 125 70 The average MFI increased 1.5-fold with knockout of the COSMC 90 70 chaperone and 133.4-fold when combined with sialidase treatment 38 50 compared to WT K562s, reflecting a high abundance of STn anti- kDa 25 gens in the COSMC KO K562 cell line (Fig. 3B). In addition, this StcEE447D Total protein kDa α-MUC1 2° only Total StcEE447D staining was highly sensitive to pretreatment with active BT4244 (SI protein Appendix,Fig.S7), further demonstrating the ability of the probe to D F detect shifts in mucin populations following perturbations targeting E326A PBS VC SiaBT+Sia PBS VC SiaBT+Sia mucins with truncated O-glycans. In contrast, AF647-AM0627 BSAfetuinC1-INHpodocalyxinMUC16PSGL-1CD45 BSAfetuinC1-INHpodocalyxinMUC16PSGL-1CD45 exhibited no binding against COSMC KO K562 cells with or without 260 sialidase treatment and was therefore not pursued further. The in- 260 ability of the AM0627 point mutant to bind to the same samples 125 125 90 despite its shared recognition of desialylated, truncated O-glycans 4of9 | www.pnas.org/cgi/doi/10.1073/pnas.2012196117 Shon et al. Downloaded by guest on October 4, 2021 slices, implying that the point mutant was recognizing the same derived from biotin-StcEE447D binding represented a subset of the pool of epitopes cleaved by the active enzyme instead of binding AB/PAS signal (Fig. 4B and SI Appendix,Fig.S12) and was able to nonspecifically (SI Appendix, Fig. S11). The StcE pretreatment clearly distinguish disparate mucin levels between diseased and did not, however, remove all of the signal, likely from an inability healthy tissues. For example, mucin-producing gastric signet ring to release cross-linked mucins from the fixed tissue and/or break cell carcinoma (38) exhibited significantly greater staining by interactions involved in mucin packing within goblet cells that StcEE447D compared to gastric adenocarcinoma and healthy gas- lie outside of the mucin domain (37). To further compare the tric tissue whereas AB/PAS staining intensities did not differ ap- selectivity of biotin-StcEE447D to that of AB/PAS, we stained a preciably between the three cases (Fig. 4C). The same was true microarray consisting of both normal and cancerous tissues. Staining when comparing mucinous histological subtypes of ovarian and A StcEE447D AB/PAS 2° only Jejunum Jejunum Jejunum B StcEE447D StcEE447D StcEE447D Muscularis externa Myometrium Salivary gland AB/PAS AB/PAS AB/PAS BIOCHEMISTRY Muscularis externa Myometrium Salivary gland C StcEE447D StcEE447D StcEE447D Normal stomach Adenocarcinoma Signet ring cell carcinoma AB/PAS AB/PAS AB/PAS Normal stomach Adenocarcinoma Signet ring cell carcinoma D BT4244E575A FOV1 BT4244E575A FOV2 BT4244E575A FOV3 VC Sia-treated jejunum VC Sia-treated jejunum VC Sia-treated jejunum 50 μm Fig. 4. Mucinase point mutants visualize mucin-domain glycoproteins in tissues. (A, Left) Immunohistochemistry (IHC) of small intestine jejunum tissue with biotin-StcEE447D (20 μg/mL) visualized by 3,3′-diaminobenzidine (DAB) staining (brown). A mucin-filled goblet cell and the mucus layer are denoted by the red and blue arrows, respectively. (A, Center) Staining of small intestine jejunum tissue with AB (dark purple)/PAS (pink, magenta). Cytoplasmic PAS staining is shown by the green arrow. (A, Right) Secondary (2°) only control staining of small intestine jejunum tissue with streptavidin-HRP and DAB. (B, Top row) Biotin- StcEE447D IHC staining and (B, Bottom row) AB/PAS staining of small intestine muscularis externa, myometrium, and salivary gland tissues. (C, Top row) Biotin- StcEE447D IHC staining and (C, Bottom row) AB/PAS staining of normal stomach, gastric adenocarcinoma, and gastric signet ring cell carcinoma tissues. (D)IHC staining of small intestine jejunum tissue, incubated with 10 nM VC Sia for 2 h at 37 °C in PBS, with biotin-BT4244E575A (10 μg/mL) visualized by DAB. Each panel shows a different field of view (FOV). All tissues were counterstained with hematoxylin (blue) and imaged using a Leica DM2000 microscope. (Scale bars: A–D, 100 μm; D, Right,50μm.) Shon et al. PNAS Latest Articles | 5of9 Downloaded by guest on October 4, 2021 lung cancer compared to other subtypes that are less associated biomarkers in disease. Additionally, they motivate continued with mucin production (SI Appendix,Fig.S13)(39–41). Thus, efforts to develop mass spectrometry techniques that would biotin-StcEE447D is a reliable tool for probing total mucin enable full sequence and glycoform mapping of patient- composition in tissues. derived CA125. Biotin-BT4244E575A detected a subset of goblet cell mucin granules compared to biotin-StcEE447D, and only in sialidase- Discussion pretreated small intestine jejunum slices (Fig. 4D), possibly We have identified and characterized a collection of mucin- reflecting an inaccessibility or lack of truncated glycoforms in selective bacterial proteases with unique peptide and glycan secreted mucins. Staining with biotin-Vicia Villosa Lectin (VVL), specificities, expanding the available research toolkit for mucin which recognizes α-orβ-linked terminal GalNAc residues, showed proteolysis. These enzymes span four distinct MEROPS pepti- higher-intensity staining of goblet cells and greater cytoplasmic dase families, including both serine proteases and zinc metal- staining, perhaps resulting from its lack of specificity for mucin loproteases. All of the tested candidates were capable of cleaving substrates (SI Appendix,Fig.S14). As with biotin-StcEE447D,the recombinant mucin-domain glycoproteins CD43, PSGL-1, MUC16, signal derived from biotin-BT4244E575A binding was sensitive to and podocalyxin, with the majority exhibiting selectivity for mucin- active BT4244 pretreatment (SI Appendix,Fig.S15). Biotin- domain glycoprotein C1-INH over fetuin, which is O-glycosylated BT4244E575A is therefore a more selective tissue-staining probe but is not a mucin. Manual mapping of cleavage sites via mass for mucins containing truncated, desialylated O-glycans. spectrometry revealed the minimal peptide and glycan require- ments for recognition by each mucinase and their sensitivities to Mucinase Toolkit Enables Patient Grouping. To explore the utility of the presence of sialic acid. To note, these particular mucinases the mucinase toolkit in assessing clinically relevant mucins, we were unique in their requirement for mucin-type O-glycosylation. probed CA125/MUC16 glycosylation and mucin expression in ToxR-activated gene A (TagA) from V. cholerae was previously samples from ovarian cancer patients. By screening activity of reported to exhibit selective activity for mucin substrates and the glycosylation-sensitive mucinase panel against CA125 in as- shares the highest sequence homology (37%) with StcE (42). cites fluid from patients, we found that treatment with AM0627 However, mass spectrometry characterization of purified TagA in combination with VC sialidase resulted in unique cleavage revealed that mucin-type O-glycosylation is not required for patterns between samples that could be separated into three cleavage (SI Appendix,Fig.S18and Dataset S7). Thus, classifi- distinct groups (Fig. 5A and SI Appendix, Fig. S16), potentially cation into the four MEROPS peptidase families highlighted in reflecting differences in CA125 proteoforms and glycoforms. this work does not necessarily guarantee mucinase activity, which The bands observed were not generated by proteases naturally likely involves structural elements that are not obvious based on present in the samples (SI Appendix,Fig.S17) and were unique primary amino acid sequence. from the patterns obtained by digesting MUC16 in healthy We further engineered two of these mucinases to be inactive cervix tissue from three individuals. In addition, immunohis- and demonstrated their application as staining reagents for the tochemistry staining with biotin-StcEE447D of grade 2 mucin- detection of mucins in a glycoform-selective manner on live cell ous papillary ovarian adenocarcinoma tissues from six patients surfaces, membrane-immobilized proteins, and tissue slices. revealed subtle differences in mucin staining intensity and Specifically, the catalytic point mutant of StcE acts as a pan-mucin distribution among patients (Fig. 5B). Assessment of the binding agent that simultaneously recognizes a wide range of clinical relevance of these findings requires many more sam- glycans in addition to the unique mucin secondary structure, as ples from patients. Nevertheless, these experiments serve to corroborated by molecular modeling with a previously described underscore the potential applications of the mucinase toolkit crystal structure (33). We showed that this point mutant requires for studying glycosylation and expression patterns of mucin GalNAc-initiated mucin-type O-glycosylation for binding and is ABHealthy cervix lysate Ovarian cancer patient ascites Grade 2 mucinous papillary ovarian adenocarcinoma ABCDEFGHI JKLMNOPQRST StcEE447D StcEE447D StcEE447D 260 125 70 kDa AM0627+VC Sia-treated α-MUC16 StcEE447D StcEE447D StcEE447D BFIGCM DHJPQR S 260 125 70 kDa Fig. 5. Mucinase toolkit enables characterization and binning of patients. (A) Ascites fluid from ovarian cancer patients and cervix tissue lysate containing 20 U of CA125 (MUC16) were treated with 200 nM AM0627 and 10 nM VC Sia for 1 h at 37 °C. Western blot with anti-MUC16 antibody (X75) was performed, and patient samples were grouped based on resulting cleavage patterns. (B) IHC staining of grade 2 mucinous papillary ovarian adenocarcinoma tissues with biotin-StcEE447D (20 μg/mL) visualized by DAB. Tissues were counterstained with hematoxylin (blue) and imaged using a Leica DM2000 microscope. (Scale bar, 100 μm.) 6of9 | www.pnas.org/cgi/doi/10.1073/pnas.2012196117 Shon et al. Downloaded by guest on October 4, 2021 sensitive to pretreatment with mucin-selective proteases. As a therapeutically target the relationships observed between altered staining reagent, StcEE447D can reliably detect changes in global mucin mucin glycosylation and disease. composition in various cell lines, lysates, and tissues by flow cytom- etry, Western blot, and immunohistochemistry. Materials and Methods In contrast, the catalytic point mutant of BT4244 strongly Materials. Recombinantly expressed podocalyxin, MUC16, PSGL-1, CD43, and prefers desialylated and truncated O-glycans, particularly the Tn CD45 were purchased from R&D Systems (1658-PD, 5609-MU, 3345-PS, antigen, as explained by the crystal structure (22). We demon- 9680-CD, and 1430-CD, respectively). C1-INH purified from human plasma strated that this probe is also capable of detecting changes in was purchased from Molecular Innovations (HC1INH). BSA was purchased from Sigma-Aldrich (A7906-1KG). Fetuin was purchased from Promega mucin levels in various samples by flow cytometry, Western blot, (V4961). Small Intestine Jejunum Tissue Slides (Normal), the Human Multi- and immunohistochemistry. However, the mucin selectivity tissue Tissue Microarray (Cancer), and Human Cervix Whole Tissue Lysate E575A combined with the restricted glycan requirement of BT4244 were purchased from Novus Biologicals (NBP2-30201, NBP2-30263, NB820- can lower the sensitivity of this reagent in certain situations. In 59275). A multiple ovarian carcinoma tissue microarray was purchased from complex samples, such as cell lysates or tissues, truncated US Biomax, Inc. (OV483). Deidentified clinical samples from consenting pa- O-glycans within densely glycosylated mucin domains may not be tients were obtained from the Department of Obstetrics and Gynecology, easily accessible for a relatively large mucinase like BT4244, Stanford Hospital. especially when surrounded by more elongated glycan structures. Thus, detection by the probe will depend on how the recognized In Vitro Mucinase Activity Assays. Digests against IRDye 800CW-C1-INH E575A (1.90 mol dye/mol protein) and IRDye 800CW-fetuin (1.67 mol dye/mol epitope is displayed. This may explain why biotin-BT4244 ± D protein) were performed at a 1:50 E:S ratio (40 nM mucinase) 10 nM VC Sia detected only PSGL-1 by Western blot (Fig. 3 ), despite being in phosphate-buffered saline (PBS) (9 μL of total volume) for 16 h at 37 °C. able to cleave all of the mucin substrates. It is likely the case that Reactions were loaded onto a 4 to 12% Criterion XT Bis-Tris precast gel the other membrane-immobilized mucins did not present motifs (Bio-Rad) and run in XT-MOPS (Bio-Rad) at 180 V for 1 h. Gels were imaged recognized by BT4244 in an accessible manner. We believe this is using an Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR less of an issue for StcE because it can tolerate a much larger Biosciences). variety of glycan structures. However, under the right conditions, the BT4244E575A probe is capable of producing robust signal, as MS Characterization of Mucinase Cleavage Activity. A protocol for sample shown by the large shifts in binding by flow cytometry with in- preparation is provided in SI Appendix. Samples were analyzed by online B nanoflow liquid chromatography-tandem mass spectrometry using an creased presentation of the Tn antigen (Fig. 3 ). Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled Interestingly, the Tn, STn, and T antigens are often up- to a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific). A portion of the BIOCHEMISTRY regulated on mucin substrates in cancer and are thought to sample (4 μLof10μL; 40%) was loaded via autosampler isocratically onto a promote metastasis (43, 44). As a result, therapeutic targeting of C18 nano precolumn using 0.1% formic acid in water (“solvent A”). For cancer-associated glycoforms of mucins, such as MUC1-Tn, is an preconcentration and desalting, the column was washed with 2% acetoni- active area of research (45, 46). We envision that the tools trile and 0.1% formic acid in water (“loading pump solvent”). Subsequently, presented here will enable the discovery of additional mucin the C18 nano precolumn was switched in line with the C18 nano separation signatures with associated alterations in glycosylation pattern column (75-μm × 250-mm EASYSpray containing 2 μm C18 beads) for gra- that contribute to disease. As shown in our proof-of-concept dient elution. The column was held at 40 °C using a column heater in the EASY-Spray ionization source (Thermo Fisher Scientific). The samples were experiments, these reagents are capable of characterizing and eluted at a constant flow rate of 0.3 μL/min using a 90-min gradient. The binning patients based on disparate mucin expression levels and gradient profile was as follows (min:% solvent B, 2% formic acid in aceto- glycosylation patterns. Identification of the differences in mucin nitrile): 0:3, 3:3, 93:35, 103:42, 104:95, 109:95, 110:3, 140:3. The instrument composition and glycosylation between patient groups may re- method used an MS1 resolution of 60,000 full width at half maximum veal specific mucin markers involved in immune regulation that (FWHM) at 400 m/z, an automatic gain control (AGC) target of 3e5, and a correlate with clinical outcomes. For example, the glycoimmune mass range from 300 to 1,500 m/z. Dynamic exclusion was enabled with a checkpoint receptor sialic acid-binding Ig-type lectin-7 (Siglec-7) repeat count of 3, repeat duration of 10 s, and exclusion duration of 10 s. has been shown to bind sialomucins as ligands (15, 47). Identi- Only charge states 2 to 6 were selected for fragmentation. MS2s were fying and understanding the interactions between Siglec-7 and its generated at top speed for 3 s. Higher-energy collisional dissociation (HCD) was performed on all selected precursor masses with the following param- ligands thus require mucin-selective tools. eters: isolation window of 2 m/z, 30% collision energy, orbitrap detection In the case of ovarian cancer, previous work suggests that (resolution of 30,000), and an AGC target of 1e4 ions. Electron-transfer CA125 interacts with Siglec-9 to cause systemic immune sup- dissociation (ETD) was performed if 1) the precursor mass was between pression that promotes growth and metastasis of the tumor (48). 300 and 1,000 m/z and 2) 3 of 9 HexNAc or NeuAc fingerprint ions (126.055, However, the clinical CA125 serum assay used to monitor dis- 138.055, 144.07, 168.065, 186.076, 204.086, 274.092, and 292.103) were ease progression is not predictive of clinical outcome (49). The present at ± 0.1 m/z and greater than 5% relative intensity. ETD parameters limitations of this assay have been attributed to an incomplete were as follows: calibrated charge-dependent ETD times, 2e5 reagent tar- understanding of CA125 at a molecular level and the fact that get, and precursor AGC target 1e4. MS data analysis is described in SI Appendix. antibodies used for detection are agnostic to glycosylation (50). Thus, tools for probing the glycosylation landscape of CA125 Flow Cytometry with Mucinase Stains. GALE KO K562s (5 × 105 cells) were could lead to detection reagents that correlate better with clin- cultured in the presence of media alone, 10 μM Gal, 100 μM GalNAc, or ical outcome. Stratification of patients based on CA125 glyco- 10 μM Gal and 100 μM GalNAc for 48 h. Cells were washed twice in PBS and sylation using the tools presented here may greatly aid the effort transferred in PBS to a 96-well, V-bottom plate. Cells were spun down at toward improved diagnostics. 550 × g for 5 min, resuspended in 5 μg/mL AF647-StcEE447D (4.39 mol dye/mol Finally, the unique molecular surfaces formed by clustered E447D) in cold PBS with 0.5% BSA, and incubated for 1 h at 4 °C. Following glycan patches within extended mucin domains may be involved three washes with cold PBS with 0.5% BSA and 2 mM ethylenediaminetetra- in binding interactions that have yet to be discovered. These acetic acid (EDTA), cells were stained with Sytox Green Nucleic Acid Stain patches will be defined both by the specific glycan structures and (Thermo Fisher Scientific), according to manufacturer recommendations, prior to live cell flow cytometry. by their arrangement. The molecular details of these domains K562s and COSMC KO K562s (5 × 105 cells) were washed once with PBS are required to understand their recognition by bacteria colo- and then treated with 10 nM VC Sia in 0.5 mL of Hank’s buffered salt so- nizing host mucosal layers and their roles in immune signaling. lution (HBSS) for 2 h at 37 °C. Cells were washed twice with PBS, resuspended We envision that further structure-guided engineering of en- in 10 μg/mL AF647-BT4244E575A (6.12 mol dye/mol E575A) in cold PBS with zymes to target saccharide patches will aid efforts to explain and 0.5% BSA, and incubated for 1 h at 4 °C. Washes and cell viability staining Shon et al. PNAS Latest Articles | 7of9 Downloaded by guest on October 4, 2021 were performed as described above. All flow cytometry data were analyzed in PBS. For mucinase staining, endogenous peroxidases were quenched for using FlowJo v. 10.0 (TreeStar). 10 min at room temperature in Hydrogen Peroxide Blocking Reagent (Abcam). Slides were washed in PBS-T (0.05% Tween) and incubated in Western Blotting with Mucinase Stains. Each substrate (1 μg) was loaded onto Carbo-Free Blocking Solution for 30 min. Biotin-StcEE447D (20 μg/mL) or a 4 to 12% Criterion XT Bis-Tris precast gel and run in XT-MOPS at 180 V for biotin-BT4244E575A (10 μg/mL) was applied to slides in PBS for 30 min. Fol- 1 h. The gel was transferred to a 0.2-μm nitrocellulose membrane using the lowing two washes with PBS-T, tissues were incubated with streptavidin-HRP Trans-Blot Turbo Transfer System (Bio-Rad) at 2.5 A for 15 min. Total protein (Abcam) (1:10,000) in PBS for 30 to 60 min. Slides were washed three times was quantified using REVERT stain (LI-COR Biosciences). The membrane was with PBS-T and mucinase substrates were visualized using the DAB Substrate blocked with Carbo-free Blocking Solution (Vector Laboratories) for 1 h at Kit (Abcam) according to manufacturer recommendations. Tissues were room temperature and then incubated with 5 μg/mL biotin-StcEE447D counterstained with hematoxylin (Abcam); dehydrated in a 75%, 80%, 95%, (1.89 mol biotin/mol E447D) in PBS-T (0.1% Tween) at room temperature for 100% ethanol gradient (2 × 3 min each); and cleared in xylene (4 × 5 min). 1 h. IRDye 800CW streptavidin (LI-COR Biosciences) was used according to Cover slides were mounted with Vectamount Permanent Mounting Medium manufacturer recommendations. For BT4244E575A staining, glycoproteins (Vector Laboratories) prior to imaging using a Leica DM2000 microscope. were treated for 1 h with 10 nM VC Sia at 37 °C in PBS. Primary staining was performed with 5 μg/mL biotin-BT4244E575A (1.37 mol biotin/mol E575A) in Mucinase Treatment of Patient-Derived CA125 (MUC16). Frozen ascites fluid PBS-T (0.2% Tween). derived from ovarian cancer patients and cervix tissue lysate were thawed at 6 K562s (2 × 10 cells) were washed once with PBS and then incubated with room temperature and then centrifuged at 500 × g for 5 min at 4 °C. CA125 PBS or 100 nM StcE for 1.5 h at 37 °C in 1 mL of HBSS. Treated cells were spun concentration was measured using a Human CA125 ELISA Kit (Abcam) down at 300 × g for 5 min, washed twice with PBS, and lysed in 200 μLof according to manufacturer recommendations. A portion of the clarified xTractor buffer (Clontech) supplemented with 2 μL of 100× EDTA-free pro- solution equivalent to 20 U was treated with 200 nM AM0627 and 10 nM VC tease inhibitor and 10 U of DNase I. A portion of the clarified lysate (15 μL) Sia for 1 h at 37 °C. Reactions were loaded onto a 3 to 8% Criterion XT Tris-Acetate was loaded onto a 4 to 12% Criterion XT Bis-Tris precast gel and run in XT- precast gel (Bio-Rad) and run in XT-Tricine (Bio-Rad) at 150 V for 80 min. The MOPS at 180 V for 75 min. The gel was transferred and stained as described gel was transferred to a 0.2-μm nitrocellulose membrane using the Trans- E447D above with 10 μg/mL biotin-StcE .MUC1stainingwasperformedusingan Blot Turbo Transfer System at 2.5 A for 15 min. Total protein was quantified anti-MUC1 antibody (214D4) (EMD Millipore) and IRDye 800CW Goat anti- using REVERT stain. The membrane was blocked with 5% milk in PBS-T Mouse IgG (LI-COR Biosciences) according to manufacturer recommendations. (0.1% Tween) for 1 h at room temperature and then stained using an anti- 6 COSMC KO K562s (2 × 10 cells) were spun down, washed twice with PBS, MUC16 antibody (X75) (Abcam) and IRDye 800CW Goat anti-Mouse IgG and lysed in 200 μL of xTractor buffer supplemented with 2 μL of 100× EDTA- according to manufacturer recommendations. free protease inhibitor and 10 U of DNase I. The clarified lysate (12 μL) was incubated with PBS, 10 nM VC Sia, or 10 nM VC Sia and 100 nM BT4244 for Data Availability. All study data are included in this article and SI Appendix. 1.5 h at 37 °C. Reactions were loaded onto a 4 to 12% Criterion XT Bis-Tris precast gel and run in XT-MOPS at 180 V for 75 min. The gel was transferred ACKNOWLEDGMENTS. We thank Natalie Strynadka (University of British as described above, blocked with Odyssey blocking buffer (PBS) (LI-COR Columbia), Robert Hirt (Newcastle University), James Nataro (University of μ Biosciences) for 1 h at room temperature, and stained with 5 g/mL biotin- Maryland School of Medicine), and Rodney Welch (University of Wisconsin- E575A BT4244 for 1 h at room temperature in PBS-T (0.2% Tween). IRDye Madison) for their gifts of the StcE, BT4244, Pic, and TagA expression 800CW streptavidin was used according to manufacturer recommendations. plasmids, respectively. We thank the Stanford Cell Sciences Imaging Facility for access to a Leica DM2000 microscope. This work was supported, in part, Immunohistochemistry with Mucinase Stains. Tissues were dried in an oven at by National Cancer Institute Grant R01CA200423 (to C.R.B.). D.J.S. was 62 °C for 1 h. Slides were dewaxed in xylene (4 × 5 min) and then hydrated in supported by a National Science Foundation Graduate Research Fellowship and Stanford Graduate Fellowship. S.A.M. was supported by a National a 100%, 95%, 75% ethanol gradient (2 × 3 min each). Antigen retrieval was Institute of General Medical Sciences F32 Postdoctoral Fellowship (F32- performed in Antigen Unmasking Solution, Citric Acid Based (Vector Labo- GM126663-01). K.P. was supported by a National Science Foundation ratories) for 15 min. AB/PAS staining was completed using the Alcian Blue Graduate Research Fellowship, a Stanford Graduate Fellowship, and the PAS Stain Kit (Abcam) according to manufacturer instructions. Sialidase Stanford Chemistry, Engineering & Medicine for Human Health (ChEM-H) treatment of tissue slides with 10 nM VC Sia was performed for 2 h at 37 °C Chemistry/Biology Interface Predoctoral Training Program. 1. C. E. Wagner, K. M. Wheeler, K. Ribbeck, Mucins and their role in shaping the func- 16. 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