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O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

Tumor-associated glycoproteins are a group of antigens with high immunogenic interest: The glycoforms generated by the aberrant glycosylation are tumor-specific and the novel glycoepitopes exposed can be targets of tumor-specific immune responses. The MUC1 antigen is one of the most relevant tumor-associated glycoproteins. In cancer, MUC1 loses polarity and becomes overexpressed and hypoglycosylated. Changes in glycan moieties contribute to MUC1 immunogenicity and can modify the interactions of tumor cells with antigen-presenting cells such as dendritic cells that would affect the overall antitumor immune response. Here, we show that the form of the MUC1 antigen, i.e., soluble or as microvesicle cargo, influences MUC1 processing in dendritic cells. In fact, MUC1 carried by microvesicles translocates from the endolysosomal/HLA-II to the HLA-I compartment and is presented by dendritic cells to MUC1-specific CD8+ T cells stimulating IFN-γ responses, whereas the soluble MUC1 is retained in the endolysosomal/HLA-II compartment independently by the glycan moieties and by the modality of internalization (receptor-mediated or non–receptor mediated). MUC1 translocation to the HLA-I compartment is accompanied by deglycosylation that generates novel MUC1 glycoepitopes. Microvesicle-mediated transfer of tumor-associated glycoproteins to dendritic cells may be a relevant biologic mechanism in vivo contributing to define the type of immunogenicity elicited. Furthermore, these results have important implications for the design of glycoprotein-based immunogens for cancer immunotherapy. Cancer Immunol Res; 2(2); 177–86. ©2013 AACR.

Cellular distribution of HLAII-DR molecules in iDCs was visualized after 2 or 12 hours from cell harvesting and incubation at 37{degree sign}C (columns 1 and 2, respectively) by immunofluorescence staining by Apotome module employing the anti-HLA-DR MoAb L243 (red signal). Nuclei are visualized in blu DAPI staining. Magnification 60X; Bars: 10μm.

Cellular distribution of HLAII-DR molecules in iDCs was visualized after 2 or 12 hours from cell harvesting and incubation at 37{degree sign}C (columns 1 and 2, respectively) by immunofluorescence staining by Apotome module employing the anti-HLA-DR MoAb L243 (red signal). Nuclei are visualized in blu DAPI staining. Magnification 60X; Bars: 10microm.

A, MUC1 glycoform expression of MUC1-DG75 transfectant detected by flow cytometry employing anti-MUC1 glycoform specific MoAbs. Only the ST-MUC1 glycoform, (MoAb MYE12), is expressed on the surface of transfectant cells. B, MUC1 distribution on MVs shed by MUC1-DG75 cells as detected by Immunogold staining in TEM using the anti-MUC1 MoAb Ma552. C, MUC1 detection in cell extracts and MVs of MUC1-DG75 and DG75 cell lines by Western Blot analysis employing the anti-MUC1 MoAb Ma552. The specific bands above 170kDa are present only in the MUC1-DG75 cell extract and MVs. D, ELISA of the MUC1 glycoprofile expressed by MUC1-DG75-MVs and by rST-MUC1. The ST-MUC1 is the unique MUC1 glycoepitope detected in both samples.

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PDF file - 248K, Supplementary Figure 1. (A) Simplified schematic of mucin type O-glycans analyzed in this report. Mucin-type O-linked glycans are initiated by the addition of an N-acetyl galactose sugar residue (the Tn epitope), which can be extended into the T antigen or core 3 structures, or Tn can be sialylated creating terminal STn that cannot be further extended. T antigen can be extended into core 2 structures that lead to the Lewis Blood Group antigens - LeX, SLeX, SLeC and sialyl Lewis A (the CA19-9 antigen, a widely utilized biomarker of adenocarcinoma progression). B & C - Representative immunohistochemical results for comparison of expression of mucin core proteins, glycans, and glycopeptides taken at 200x magnification. (B) Serial sections of primary tumor from autopsy patient 3 stained for indicated antigens. (C) Serial sections of liver metastasis from autopsy patient 20 for same antigens seen in primary tumor.

Purpose: Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed in pancreatic adenocarcinoma—sialyl Tn (STn), Tn, T antigen, sialyl Lewis A (CA19-9), sialyl Lewis C (SLeC), Lewis X (LeX), and sialyl LeX (SLeX)—during the progression of pancreatic cancer from early stages to metastatic disease.Experimental Design: Immunohistochemical analyses of mucin and associated glycan expression on primary tumor and liver metastatic tumor samples were conducted with matched sets of tissues from 40 autopsy patients diagnosed with pancreatic adenocarcinoma, 14 surgically resected tissue samples, and 8 normal pancreata.Results: There were significant changes in mucin expression patterns throughout disease progression. MUC1 and MUC4 were differentially glycosylated as the disease progressed from early pancreatic intraepithelial neoplasias to metastatic disease. De novo expression of several mucins correlated with increased metastasis indicating a potentially more invasive phenotype, and we show the expression of MUC6 in acinar cells undergoing acinar to ductal metaplasia. A “cancer field-effect” that included changes in mucin protein expression and glycosylation in the adjacent normal pancreas was also seen.Conclusions: There are significant alterations in mucin expression and posttranslational processing during progression of pancreatic cancer from early lesions to metastasis. The results are presented in the context of how mucins influence the biology of tumor cells and their microenvironment during progression of pancreatic cancer. Clin Cancer Res; 19(8); 1981–93. ©2013 AACR.

PDF file - 320K, Supplementary Figure 2. Representative immunohistochemical results for comparison of cancer fieldeffects in autopsy and resection tissue samples from the same patients. Two different autopsy patients who underwent surgical resection are presented. Serial sections stained for the antigens indicated are shown in the resection tissue samples alongside the autopsy samples of their primary tumors. 200x magnification.

Purpose: Adhesion of disseminating tumor cells to the blood vascular endothelium is a pivotal step in metastasis. Previous investigations have shown that galectin-3 concentrations are increased in the bloodstream of patients with cancer and that galectin-3 promotes adhesion of disseminating tumor cells to vascular endothelium in vitro and experimental metastasis in vivo. This study determined the levels of galectin-1, -2, -3, -4, -8, and -9 in the sera of healthy people and patients with colon and breast cancer and assessed the influence of these galectins on cancer-endothelium adhesion.Experimental Design: Serum galectins and auto–anti-MUC1 antibodies were assessed using ELISA and mucin protein (MUC1) glycan microarrays, and cancer-endothelium adhesion was determined using monolayers of human microvascular lung endothelial cells.Results: The levels of serum galectin-2, -3, -4, and -8 were significantly increased up to 31-fold in patients with cancer and, in particular, those with metastases. As previously shown for galectin-3, the presence of these galectins enhances cancer-endothelium adhesion by interaction with the Thomsen-Friedenreich (TF; Galβ1,3GalNAcα-) disaccharide on cancer-associated MUC1. This causes MUC1 cell surface polarization, thus exposing underlying adhesion molecules that promote cancer-endothelium adhesion. Elevated circulating galectin-2 levels were associated with increased mortality in patients with colorectal cancer, but this association was suppressed when anti-MUC1 antibodies with specificity for the TF epitope of MUC1 were also present in the circulation.Conclusions: Increased circulation of several members of the galectin family is common in patients with cancer and these may, like circulating galectin-3, also be involved in metastasis promotion. Clin Cancer Res; 17(22); 7035–46. ©2011 AACR.

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Supplementary Results, Figures 1-2, Table 1 from Cancer Biomarkers Defined by Autoantibody Signatures to Aberrant O-Glycopeptide Epitopes

In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are up-regulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in up-regulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as down-regulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

Transforming growth factor–β (TGF-β) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-β signaling pathway, including the ligands TGF-β1, TGF-β2, and TGF-β3, the receptor TGF-βRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-β signaling, the loss of TGF-β1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-βRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-βRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-βRII. This study provides a framework for exploring TGF-β signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generated a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures had minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affected glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3β oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs.

SummaryEbola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. Using glycoengineered cell lines we demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. Next, using TMT isobaric labelling, we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, as well as compared it to patterns on wild type virus-like particles. We demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. This work represents a comprehensive site-specific analysis of EBOV GP, with 47 O-glycosites identified, and sheds light on differential regulation of EBOV GP glycosylation initiated by host GalNAc-Ts. Together with the effect on viral propagation it opens prospective avenues for tailored intervention approaches and means for modulating immunogen O-glycan density.

The family of galectins has critical functions in a wide range of biological processes, primarily based on their broad interactions with proteins carrying β-galactoside-containing glycans. To understand the diversity of functions governed by galectins, it is essential to define the binding specificity of the carbohydrate recognition domain (CRDs) of the individual galectins. The binding specificity of galectins has primarily been examined with glycoarrays, but now the ability to probe and dissect binding to defined glycans in the context of a cellular membrane is facilitated by the generations of glycoengineered cell libraries with defined glyco-phenotypes. The following section will show how galectin specificities can be probed in the natural context of cellular surfaces using glycoengineered cell libraries, and how binding to glycoproteins can be measured in solution with fluorescence anisotropy.

The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens also found in healthy tissues. Such targets often do not display sufficient cancer selectivity to be used for potent antibody based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in the expression of tumor-associated carbohydrate antigens that are not present on healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumours, but only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and through a glycopeptide immunization strategy developed a cancer-specific monoclonal antibody, 4C8. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer selective by immunohistochemistry of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression, and increased survival in vivo. Importantly, 4C8 CAR T cells selectively killed target cells in a mixed organotypic skin cancer model without affecting healthy CD44v6+ keratinocytes, indicating tolerability and safety. Targeting aberrantly glycosylated glycopeptide epitopes may provide the cancer selectivity needed for solid tumour immunotherapy. Citation Format: Mikkel K.M. Aasted, Aaron C. Groen, John T. Keane, Sally Dabelsteen, Edwin Tan, Julia Schnabel, Fang Liu, Hyeon-Gyu S. Lewis, Constantine Theodoropulos, Avery D. Posey, Hans H. Wandall. Targeting solid cancers with a cancer-specific monoclonal antibody to surface expressed aberrant O-glycosylated proteins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5659.

Background Novel immunotherapies targeting cancer-associated truncated O-glycans Tn (GalNAcα-Ser/Thr) and STn (Neu5Acα2-6GalNacα-Ser/Thr) are promising strategies for cancer treatment. However, no comprehensive, antibody-based mapping of truncated O-glycans in tumours exist to guide drug development. Methods We used monoclonal antibodies to map the expression of truncated O-glycans in >700 tissue cores representing healthy and tumour tissues originating from breast, colon, lung, pancreas, skin, CNS and mesenchymal tissue. Patient-derived xenografts were used to evaluate Tn expression upon tumour engraftment. Results The Tn-antigen was highly expressed in breast (57%, n = 64), colorectal (51%, n = 140) and pancreatic (53%, n = 108) tumours, while STn was mainly observed in colorectal (80%, n = 140) and pancreatic (56%, n = 108) tumours. We observed no truncated O-glycans in mesenchymal tumours (n = 32) and low expression of Tn (5%, n = 87) and STn (1%, n = 75) in CNS tumours. No Tn-antigen was found in normal tissue (n = 124) while STn was occasionally observed in healthy gastrointestinal tissue. Surface expression of Tn-antigen was identified across several cancers. Tn and STn expression decreased with tumour grade, but not with cancer stage. Numerous xenografts maintained Tn expression. Conclusions Surface expression of truncated O-glycans is limited to cancers of epithelial origin, making Tn and STn attractive immunological targets in the treatment of human carcinomas.

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.

Mucin type O-glycosylation is one of the most diverse types of glycosylation, playing essential roles in tissue development and homeostasis. In complex organisms, O-GalNAc glycans comprise a substantial proportion of the glycocalyx, with defined functions in hemostatic, gastrointestinal, and respiratory systems. Furthermore, O-GalNAc glycans are important players in host-microbe interactions, and changes in O-glycan composition are associated with certain diseases and metabolic conditions, which in some instances can be used for diagnosis or therapeutic intervention. Breakthroughs in O-glycobiology have gone hand in hand with the development of new technologies, such as advancements in mass spectrometry, as well as facilitation of genetic engineering in mammalian cell lines. High-throughput O-glycoproteomics have enabled us to draw a comprehensive map of O-glycosylation, and mining this information has supported the definition and confirmation of functions related to site-specific O-glycans. This includes protection from proteolytic cleavage, as well as modulation of binding affinity or receptor function. Yet, there is still much to discover, and among the important next challenges will be to define the context-dependent functions of O-glycans in different stages of cellular differentiation, cellular metabolism, host-microbiome interactions, and in disease. In this review, we present the achievements and the promises in O-GalNAc glycobiology driven by technological advances in analytical methods, genetic engineering, and systems biology.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal. MUC4 (mucin4) is a heavily glycosylated protein aberrantly expressed in PDAC and promotes tumorigenesis via an unknown mechanism. To assess this, we genetically knocked out (KO) MUC4 in PDAC cells that did not express and did express truncated O-glycans (Tn/STn) using CRISPR/Cas9 technology. We found that MUC4 knockout cells possess less tumorigenicity in vitro and in vivo, which was further reduced in PDAC cells that express aberrant overexpression of truncated O-glycans. Also, MUC4(KO) cells showed a further reduction of epidermal growth factor receptors (ErbB) and their downstream signaling pathways in truncated O-glycan expressing PDAC cells. Tn-MUC4 specific 3B11 antibody inhibited MUC4-induced ErbB receptor and its downstream signaling cascades. MUC4 knockout differentially regulates apoptosis and cell cycle arrest in branched and truncated O-glycan expressing PDAC cells. Additionally, MUC4(KO) cells were found to be more sensitive to gemcitabine treatment. They possessed the upregulated expression of hENT1 and hCNT3 compared to parental cells, which were further affected in cells with aberrant O-glycosylation. Taken together, our results indicate that MUC4 enhances the malignant properties and gemcitabine resistance in PDAC tumors that aberrantly overexpress truncated O-glycans via altering ErbB/AKT signaling cascades and expression of nucleoside transporters, respectively.

Transforming growth factor β (TGFβ) signaling is essential for cell growth and differentiation. Yet, the role of the individual TGFβ signaling components in human tissue homeostasis and transformation is still not completely understood. Here we applied a combined strategy of genetic engineering, a human organotypic 3D skin model, and global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGFβ signaling pathway. Our results show that loss of Smad4-dependent TGFβ signaling affected skin homeostasis through regulation of ECM expression and cell cycle control. In contrast, deletion of TGFβRII resulting in the combined loss of Smad4-dependent and independent TGFβ signaling induced invasive growth through activation of p38 and ERK signaling. The study provides a framework for exploration of signaling pathways with genetic engineering, human 3D tissue models, and with global proteomics and phosphoproteomics.

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.

It has long been known that surface proteins of most enveloped viruses are covered with glycans. It has furthermore been demonstrated that glycosylation is essential for propagation and immune evasion for many viruses. The recent development of high-resolution mass spectrometry techniques has enabled identification not only of the precise structures but also the positions of such post-translational modifications on viruses, revealing substantial differences in extent of glycosylation and glycan maturation for different classes of viruses. In-depth characterization of glycosylation and other post-translational modifications of viral envelope glycoproteins is essential for rational design of vaccines and antivirals. In this Review, we provide an overview of techniques used to address viral glycosylation and summarize information on glycosylation of enveloped viruses representing ongoing public health challenges. Furthermore, we discuss how knowledge on glycosylation can be translated to means to prevent and combat viral infections.

Proteolytic processing is an irreversible post-translational modification functioning as a ubiquitous regulator of cellular activity. Protease activity is tightly regulated via control of gene expression, enzyme and substrate compartmentalization, zymogen activation, enzyme inactivation, and substrate availability. Emerging evidence suggests that proteolysis can also be regulated by substrate glycosylation and that glycosylation of individual sites on a substrate can decrease or, in rare cases, increase its sensitivity to proteolysis. Here, we investigated the relationship between site-specific, mucin-type (or GalNAc-type) O-glycosylation and proteolytic cleavage of extracellular proteins. Using in silico analysis, we found that O-glycosylation and cleavage sites are significantly associated with each other. We then used a positional proteomic strategy, terminal amine isotopic labeling of substrates (TAILS), to map the in vivo cleavage sites in HepG2 SimpleCells with and without one of the key initiating GalNAc transferases, GalNAc-T2, and after treatment with exogenous matrix metalloproteinase 9 (MMP9) or neutrophil elastase. Surprisingly, we found that loss of GalNAc-T2 not only increased cleavage, but also decreased cleavage across a broad range of other substrates, including key regulators of the protease network. We also found altered processing of several central regulators of lipid homeostasis, including apolipoprotein B and the phospholipid transfer protein, providing new clues to the previously reported link between GALNT2 and lipid homeostasis. In summary, we show that loss of GalNAc-T2 O-glycosylation leads to a general decrease in cleavage and that GalNAc-T2 O-glycosylation affects key regulators of the cellular proteolytic network, including multiple members of the serpin family.

Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome, which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.

Aberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6. GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell–cell adhesion, and formation of crypts in tissue cultures. O-Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6–specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation.

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.

BACKGROUND Genomewide association studies have reported alleles in the ABO locus to be associated with ferritin levels. These studies warrant the investigation of a possible association between the ABO blood group and ferritin levels. We aimed to explore if ABO blood group is associated with iron stores expressed as ferritin levels. STUDY DESIGN AND METHODS Ferritin levels were measured at least once for 30,595 Danish Blood Donor Study participants. Linear regression analyses were performed with the ABO blood group as explanatory variable and adjusted for age, number of donations 3 years before the ferritin measurement, and time since latest donation. In addition, a subanalysis was performed on 15,280 individuals in which further adjustments for body mass index, smoking status, and C-reactive protein levels were possible. Furthermore, logistic regression analyses were performed to determine if ABO blood group was associated with a ferritin level of less than 15 ng/mL. RESULTS Non-O blood group donors had lower ferritin levels than blood group O donors, regardless of sex. Accordingly, risk of ferritin level of less than 15 ng/mL was increased for individuals with non-O blood group compared with O blood group. In subanalyses similar associations were observed, albeit in women the association between blood group and risk of a ferritin level below 15 ng/mL was no longer significant. ABO blood group was not associated with red blood cell indices such as mean cell volume and mean cell hemoglobin content. CONCLUSION Donors with non-O blood group have lower ferritin levels than donors with other blood groups.