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Design of a mucin-selective protease for targeted degradation of cancer-associated mucins

ABSTRACT: Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide- and glycan- motif and achieve cell-type selectivity via antigen-driven cell surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in murine models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific glycoproteins on target cells.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Nicholas Riley

LAB HEAD: Carolyn R. Bertozzi

PROVIDER: PXD042243 | Pride | 2023-07-21

REPOSITORIES: Pride

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			Action	DRS
	20200824_SAM_CD43_ddStcE_EThcD.raw	Raw
	20200824_SAM_CD43_ddStcE_Sia_EThcD.raw	Raw
	20200824_SAM_MUC16_ddStcE_EThcD.raw	Raw
	20200824_SAM_MUC16_ddStcE_Sia_EThcD.raw	Raw
	20200824_SAM_PSGL1_ddStcE_EThcD.raw	Raw

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Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity

Project description:The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia (CNL). However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in CNL and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues – the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at wild-type threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain (IL-31Rα). Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.

2023-07-20 | PXD035664 | Pride

HepG2 GalNAc-T1/2 Bump-and-hole secretome glycopeptide data

Project description:Engineered GalNAc-T glycosyltransferases were used to incorporate a chemically modified GalNAC analog into the secretome of HepG2 cells. The chemical modification included a bioorthogonal alkyne tag that allowed for introduction of a clickable, acid-cleavable biotin-picolyl-azide. Glycoproteins were enriched using Streptavidin, and on-bead digestion yielded solid phase-bound glycopeptides that were released with acid. Glycopeptides were analysed.

2020-04-22 | PXD018048 | Pride

K-562 GalNAc-T1/2 Bump-and-hole Whole cell lysate proteomics data

Project description:Engineered GalNAc-T glycosyltransferases were used to incorporate a chemically modified GalNAc analog into the glycoproteins on the cell surface of K-562 cells. The chemical modification included a bioorthogonal alkyne tag that allowed for introduction of a clickable, acid-cleavable biotin-picolyl-azide. Glycoproteins were enriched using Streptavidin, and on-bead digestion yielded a peptide fraction that was analysed by mass spectrometry.

2020-04-22 | PXD017989 | Pride

Cell-type specific tagging of glycoproteins in murine-human co-culture samples glycoproteomics lysates

Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK/Nh-GalNAc-T2) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of an acid cleavable biotinylated tag. Glycoproteins are enriched using Streptavidin and. after on-bead digestion, glycopeptides are eluted in acid-conditions to be then analysed by mass spectrometry.

2022-09-10 | PXD035449 | Pride

Cell-type specific tagging of glycoproteins in murine-human co-culture secretome glycoproteomics

2022-09-10 | PXD035445 | Pride

HepG2 secretome tagged with the sugar analogs GalNAz and GalNAzMe

Project description:Chemically modified analogs of GalNAc, termed GalNAz or GalNAzMe, were incorporated in secreted glycoproteins. The compounds contain bioorthogonal, clickable functional groups that allowed for enrichment using biotin. Chemical glycoproteomics was performed after on-bead digestion and cleavage of glycopeptides from the solid support.

2020-09-14 | PXD020648 | Pride

On-tissue spatially-resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape

Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.

2022-05-04 | PXD025537 | Pride

Quantification and site-specific analysis of co-occupied N- and O-glycopeptides

Project description:Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.

2024-10-22 | PXD053713 | Pride

Exploring glycosylation of mucins with O-glycodomain reporters expressed in glycoengineered HEK293 cells

Project description:Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of resistance to proteolytic digestion, and knowledge of positions of O-glycans is particularly limited for these regions. Mucin O-glycodomains are believed to contain important binding cues for endogenous lectin receptors and the microbiota, and it is important to develop strategies to characterize and produce these abundant molecules. Here, we took advantage of our recently developed glycoengineered cell-based platform for display and production of mucin TR reporters with custom designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in MUC2, MUC20, MUC21, PSGL-1, and Syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in HEK293 SimpleCells (Tn-glycoform). Interestingly, while all the sites in TRs derived from secreted mucins were almost fully occupied, positions in TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of the StcE and BT4244 glycoproteases, revealing a more restricted substrate specificities than reported. Finally, we used these for bottom-up analysis of isolated ovine submaxillary mucin (OSM) and identified the gene. The study provides insight into O-glycosylation of mucins and mucin-like domains and the strategies developed open up for wider analysis of native mucins.

2022-05-20 | PXD029885 | Pride

4-deoxy-4-fluoro-GalNAz (4FGalNAz) is a metabolic chemical reporter of O-GlcNAc modifications, highlighting the notable substrate flexibility of O-GlcNAc transferase

Project description:Bioorthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogs of monosaccharides that contain bioorthogonal functionality, like azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bioorthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g. N-linked vs. O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat disappointing from the standpoint of a mucin-selective MCR, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

2022-02-17 | PXD027333 | Pride

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