SnapShot: O-Glycosylation Pathways across Kingdoms.

O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of O-glycans found in different organisms and their principle biosynthetic pathways. To view this SnapShot, open or download the PDF.

Molecular logic of neuronal self-recognition through protocadherin domain interactions.

Self-avoidance, a process preventing interactions of axons and dendrites from the same neuron during development, is mediated in vertebrates through the stochastic single-neuron expression of clustered protocadherin protein isoforms. Extracellular cadherin (EC) domains mediate isoform-specific homophilic binding between cells, conferring cell recognition through a poorly understood mechanism. Here, we report crystal structures for the EC1-EC3 domain regions from four protocadherin isoforms representing the α, ß, and γ subfamilies. All are rod shaped and monomeric in solution. Biophysical measurements, cell aggregation assays, and computational docking reveal that trans binding between cells depends on the EC1-EC4 domains, which interact in an antiparallel orientation. We also show that the EC6 domains are required for the formation of cis-dimers. Overall, our results are consistent with a model in which protocadherin cis-dimers engage in a head-to-tail interaction between EC1-EC4 domains from apposed cell surfaces, possibly forming a zipper-like protein assembly, and thus providing a size-dependent self-recognition mechanism.

Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase.

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C- and O-Mannosylated Peptides.

Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.

O-linked mucin-type glycosylation regulates the transcriptional programme downstream of EGFR.

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/ß-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.

Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics.

Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats.

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific ß-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1-4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific ß-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.

A strategy for generating cancer-specific monoclonal antibodies to aberrant O-glycoproteins: identification of a novel dysadherin-Tn antibody.

Successful application of potent antibody-based T-cell engaging immunotherapeutic strategies is currently limited mainly to hematological cancers. One major reason is the lack of well-characterized antigens on solid tumors with sufficient cancer specific expression. Aberrantly O-glycosylated proteins contain promising cancer-specific O-glycopeptide epitopes suitable for immunotherapeutic applications, but currently only few examples of such antibody epitopes have been identified. We previously showed that chimeric antigen receptor T-cells directed towards aberrantly O-glycosylated MUC1 can control malignant growth in a mouse model. Here, we present a discovery platform for the generation of cancer-specific monoclonal antibodies targeting aberrant O-glycoproteins. The strategy is based on cancer cell lines engineered to homogeneously express the truncated Tn O-glycoform, the so-called SimpleCells. We used SimpleCells of different cancer origin to elicit monoclonal antibodies with selectivity for aberrant O-glycoproteins. For validation we selected and characterized one monoclonal antibody (6C5) directed to a Tn-glycopeptide in dysadherin (FXYD5), known to be upregulated in cancer and promote metastasis. While dysadherin is widely expressed also in normal cells, we demonstrated that the 6C5 epitope is specifically expressed in cancer.

NleB/SseK effectors from Citrobacter rodentium, Escherichia coli, and Salmonella enterica display distinct differences in host substrate specificity.

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and Crodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2.

Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated α-dystroglycanopathies, because deficient O-Man glycosylation of α-dystroglycan disrupts laminin interaction with α-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose ß-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to α-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to α-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.

GlycoDomainViewer: a bioinformatics tool for contextual exploration of glycoproteomes.

The GlycoDomainViewer is a bioinformatic tool to aid in the mining of glycoproteomic datasets from different sources and facilitate incorporation of glycosylation into studies of protein structure and function. We present a version 2.0 of GlycoDomainViewer incorporating a number of advanced features, which enhances visibility and accessibility of the wealth of glycoproteomic data being generated. The GlycoDomainViewer enables visual exploration of glycoproteomic data, incorporating information from recent N- and O-glycoproteome studies on human and animal cell lines and some organs and body fluids. The initial data comprises sites of glycosylation for N-linked, O-GalNAc, O-Fucose, O-Xyl, O-Mannose (in both human and yeast) and cytosolic O-GlcNAc type. The data made available via this tool will be regularly updated to improve the coverage of known glycosylation sites and datasets, reflecting the advances currently being made in characterization of glycoproteomes. The tool is available at https://glycodomain.glycomics.ku.dk.

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae.

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammalsO-mannosylation is the only type ofO-glycosylation. In an essential step toward the full understanding of proteinO-mannosylation we mapped theO-mannose glycoproteome in baker's yeast. Taking advantage of anO-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500O-glycoproteins from all subcellular compartments for which over 2300O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized proteinO-mannosyltransferases. We find thatO-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed thatO-mannosylation is favored in unstructured regions and ß-strands. Furthermore,O-mannosylation is impeded in the proximity ofN-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and theirO-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types ofO-glycosylation from yeast to mammals.

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation.

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast.

Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.

Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

O-linked glycosylation of the mucin domain of the herpes simplex virus type 1-specific glycoprotein gC-1 is temporally regulated in a seed-and-spread manner.

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1-infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to Thr-87 and Thr-91 and one GalNAc unit to either Thr-99 or Thr-101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the Thr-76-Lys-107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by followup transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Because the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provide a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1-infected target cells.

Advances in mass spectrometry driven O-glycoproteomics.

BACKGROUND: Global analyses of proteins and their modifications by mass spectrometry are essential tools in cell biology and biomedical research. Analyses of glycoproteins represent particular challenges and we are only at the beginnings of the glycoproteomic era. Some of the challenges have been overcome with N-glycoproteins and proteome-wide analysis of N-glycosylation sites is accomplishable today but only by sacrificing information of structures at individual glycosites. More recently advances in analysis of O-glycoproteins have been made and proteome-wide analysis of O-glycosylation sites is becoming available as well. SCOPE OF REVIEW: Here we discuss the challenges of analysis of O-glycans and new O-glycoproteomics strategies focusing on O-GalNAc and O-Man glycoproteomes. MAJOR CONCLUSIONS: A variety of strategies are now available for proteome-wide analysis of O-glycosylation sites enabling functional studies. However, further developments are still needed for complete analysis of glycan structures at individual sites for both N- and O-glycoproteomics strategies. GENERAL SIGNIFICANCE: The advances in O-glycoproteomics have led to identification of new biological functions of O-glycosylation and a new understanding of the importance of where O-glycans are positioned on proteins.

The GalNAc-type O-Glycoproteome of CHO cells characterized by the SimpleCell strategy.

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins.

The metazoan O-mannose (O-Man) glycoproteome is largely unknown. It has been shown that up to 30% of brain O-glycans are of the O-Man type, but essentially only alpha-dystroglycan (α-DG) of the dystrophin-glycoprotein complex is well characterized as an O-Man glycoprotein. Defects in O-Man glycosylation underlie congenital muscular dystrophies and considerable efforts have been devoted to explore this O-glycoproteome without much success. Here, we used our SimpleCell strategy using nuclease-mediated gene editing of a human cell line (MDA-MB-231) to reduce the structural heterogeneity of O-Man glycans and to probe the O-Man glycoproteome. In this breast cancer cell line we found that O-Man glycosylation is primarily found on cadherins and plexins on ß-strands in extracellular cadherin and Ig-like, plexin and transcription factor domains. The positions and evolutionary conservation of O-Man glycans in cadherins suggest that they play important functional roles for this large group of cell adhesion glycoproteins, which can now be addressed. The developed O-Man SimpleCell strategy is applicable to most types of cell lines and enables proteome-wide discovery of O-Man protein glycosylation.