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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.

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.

Summary Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids. For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020)., Graphical abstract, Highlights • CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues • Approach can be used to study the role of glycans in epithelial tissue formation • Strategy applicable to other targets and organotypic tissue models, Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S – insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

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.

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.

Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N-glycosylation gene N-acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition., Graphical Abstract MGAT5 knockout (KO) in HEK293 cells induces metabolic changes resulting in increased intracellular UDP-GlcNAc, increased glycolysis, enhanced spare respiratory capacity and higher citrate flux from the mitochondria. MGAT5 KO cells express constitutively high MICA, mainly regulated on the transcriptional level through opening of the chromatin at the MICA promoter. MICA expression in MGAT5 KO cells is dependent on citrate turnover and histone acetylation. Blocking citrate flux inhibits MICA expression in numerous cancer cell lines, and we propose that this is a central metabolic regulation of MICA and immune surveillance.

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.

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.

The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play important roles in the hemostatic system; however, most investigations have focused on N-glycans because of the complexity of O-glycan analysis. Here we performed the first systematic analysis of native-O-glycosylation using lectin affinity chromatography coupled to liquid chromatography mass spectrometry (LC-MS)/MS to determine the precise location of O-glycans in human plasma, platelets, and endothelial cells, which coordinately regulate hemostasis. We identified the hitherto largest O-glycoproteome from native tissue with a total of 649 glycoproteins and 1123 nonambiguous O-glycosites, demonstrating that O-glycosylation is a ubiquitous modification of extracellular proteins. Investigation of the general properties of O-glycosylation established that it is a heterogeneous modification, frequently occurring at low density within disordered regions in a cell-dependent manner. Using an unbiased screen to identify associations between O-glycosites and protein annotations we found that O-glycans were over-represented close (± 15 amino acids) to tandem repeat regions, protease cleavage sites, within propeptides, and located on a select group of protein domains. The importance of O-glycosites in proximity to proteolytic cleavage sites was further supported by in vitro peptide assays demonstrating that proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans. Collectively, these data illustrate the global properties of native O-glycosylation and provide the requisite roadmap for future biomarker and structure-function studies.

Post‐translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O‐glycosylation is among the most abundant and diverse PTMs. Initiation of O‐GalNAc glycosylation is regulated by 20 distinct GalNAc‐transferases (GalNAc‐Ts), and deficiencies in individual GalNAc‐Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc‐Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc‐Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc‐T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc‐T1 targets are associated with components of the endomembrane system, GalNAc‐T2 targets with cell–ECM adhesion, and GalNAc‐T3 targets with epithelial differentiation. Thus, GalNAc‐T isoforms serve specific roles during human epithelial tissue formation., GalNAc‐T isoform knock out in human skin organoids reveals selective phenotypes in tissue organization and differentiation. By using mass spectrometry‐based phospho‐ and O‐glycoproteomics, the identified phenotypes are correlated with unique signaling networks and isoform‐selective protein substrates.

Summary The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models., Graphical Abstract, Highlights • Glycosphingolipids tune cell signaling and impact epithelial barrier formation • Complex N-glycans govern wound healing and lamellar body functions in human skin • Core-1 O-glycans are essential for cell-cell adhesion and human tissue differentiation • Notch O-fucosylation and O-glucosylation have distinct roles in human tissue formation, Dabelsteen et al. present a glycoengineered organotypic epithelial tissue model that allows a systematic interrogation of glycan functions in human tissue formation and homeostasis. The model shows the distinct functions of select glycoconjugates during cellular differentiation and serves as a resource for further identification of glycan molecular functions.

Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a “bottom up” mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation.

International audience; Lipids are structural components of cellular membranes and signaling molecules that are widely involved in development and diseases, but the underlying molecular mechanisms are poorly understood, partly because of the vast variety of lipid species and complexity of synthetic and turnover pathways. From a genetic screen, we identify that mannosyl glucosylceramide (MacCer), a species of glycosphingolipid (GSL), promotes synaptic bouton formation at the Drosophila neuromuscular junction (NMJ). Pharmacological and genetic analysis shows that the NMJ growth-promoting effect of MacCer depends on normal lipid rafts, which are known to be composed of sphingolipids, sterols and select proteins. MacCer positively regulates the synaptic level of Wnt1/Wingless (Wg) and facilitates presynaptic Wg signaling, whose activity is raft-dependent. Furthermore, a functional GSL-binding motif in Wg exhibiting a high affinity for MacCer is required for normal NMJ growth. These findings reveal a novel mechanism whereby the GSL MacCer promotes synaptic bouton formation via Wg signaling.

International audience; Glycosylation of proteins, lipids and proteoglycans in human cells involves at least 167 identified glycosyltransferases (GTfs), and these orchestrate the biosynthesis of diverse types of glycoconjugates and glycan structures. Mutations in this part of the genome-the GTf-genome-cause more than 58 rare, monogenic congenital disorders of glycosylation (CDGs). They are also statistically associated with a large number of complex phenotypes, diseases or predispositions to complex diseases based on Genome-Wide Association Studies (GWAS). CDGs are extremely rare and often with severe medical consequences. In contrast, GWAS are likely to identify more common genetic variations and generally involve less severe and distinct traits. We recently confirmed that structural defects in GTf genes are extremely rare, which seemed at odds with the large number of GWAS pointing to GTf-genes. To resolve this issue, we surveyed the GTf-genome for reported CDGs and GWAS candidates; we found little overlap between the two groups of genes. Moreover, GTf-genes implicated by CDG or GWAS appear to constitute different classes with respect to their: (i) predicted roles in glycosylation pathways; (ii) potential for partial redundancy by closely homologous genes; and (iii) transcriptional regulation as evaluated by RNAseq data. Our analysis suggest that more complex traits are caused by dysregulation rather than structural deficiency of GTfs, which suggests that some glycosylation reactions may be predicted to be under tight regulation for fine-tuning of important biological functions.

GalNAc‐type O‐glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O‐glycoproteome is regulated by up to 20 polypeptide GalNAc‐Ts and the contributions and biological functions of individual GalNAc‐Ts are poorly understood. Here, we used a zinc‐finger nuclease (ZFN)‐directed knockout strategy to probe the contributions of the major GalNAc‐Ts (GalNAc‐T1 and GalNAc‐T2) in liver cells and explore how the GalNAc‐T repertoire quantitatively affects the O‐glycoproteome. We demonstrate that the majority of the O‐glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non‐redundant functions of GalNAc‐T1 and GalNAc‐T2. The non‐redundant O‐glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc‐T2 isoform, these support a role in lipid metabolism. The results demonstrate that GalNAc‐Ts have different non‐redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc‐T substrates. Our study provides a new view of the differential regulation of the O‐glycoproteome, suggesting that the plurality of GalNAc‐Ts arose to regulate distinct protein functions and cellular processes.

Cutaneous T-cell lymphomas (CTCLs) are the most common primary skin lymphomas, which are characterized by an accumulation of malignant T cells in the skin. The early lesion resembles both clinically and histologically benign inflammatory disorders and also presents with hyperproliferative epidermis and T-cell infiltration. Despite considerable progress in understanding the molecular mechanisms involved in the malignant transformation of T cells, the causes of the morphological and histopathological features of the disease are largely unknown. We used an organotypic model of CTCL to show that malignant T cells through the secretion of galectin-1 and -3 stimulate vigorous growth of keratinocytes. In parallel, malignant T cells induce disorganized keratinocyte stratification, resembling the early hyperproliferative stage of CTCL. We also observed a loss of attachment between the epithelial and mesenchymal compartments. In addition, hyperproliferation was followed by a downregulation of differentiation markers, such as keratin 10 and involucrin, and a decrease in barrier formation. In conclusion, we provide evidence that malignant T cells orchestrate the histopathological epidermal changes seen in CTCL.

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

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.

Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a novel regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss-of-function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2 deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

Background Complex carbohydrates play important functions for circulation of proteins and cells. They provide protective shields and refraction from non-specific interactions with negative charges from sialic acids to enhance circulatory half-life. For recombinant protein therapeutics carbohydrates are especially important to enhance size and reduce glomerular filtration loss. Carbohydrates are, however, also ligands for a large number of carbohydrate-binding lectins exposed to the circulatory system that serve as scavenger receptors for the innate immune system, or have more specific roles in targeting of glycoproteins and cells. Scope of review Here we provide an overview of the common lectin receptors that play roles for circulating glycoproteins and cells, and present a discussion of ways to engineer glycosylation of recombinant biologics and cells to improve therapeutic effects. Major conclusions While the pharmaceutical industry has learned how to exploit carbohydrates to improve pharmacokinetic properties of recombinant therapeutics, our understanding of how to improve cell-based therapies by manipulation of complex carbohydrates is still at its infancy. Progress with the latter has recently been achieved with cold-stored platelets, where exposure of uncapped glycans lead to rapid clearance from circulation by several lectin-mediated pathways. General significance Understanding lectin-mediated clearance pathways is essential for progress in development of biological pharmaceuticals.

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions.

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.

Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post-translational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures.

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet circulation lifetime is not fully clarified. We show that thrombocytopenia in mice deficient in the St3gal4 sialyltransferase gene (St3Gal-IV−/− mice) is caused by the recognition of terminal galactose residues exposed on the platelet surface in the absence of sialylation. This results in accelerated platelet clearance by asialoglycoprotein receptor-expressing scavenger cells, a mechanism that was recently shown to induce thrombocytopenia during Streptococcus pneumoniae sepsis. We now identify platelet GPIbα as a major counterreceptor on ST3Gal-IV−/− platelets for asialoglycoprotein receptors. Moreover, we report data that establish the importance of sialylation of the von Willebrand factor in its function.

Platelet products are currently stored at room temperature, because refrigeration causes their rapid clearance from the circulation upon transfusion. Glycans have recently been emphasized as important determinants for the clearance of refrigerated platelets. The present review addresses the current knowledge of platelet glycans and the potential of glycosylation for improving platelet storage.Removal of refrigerated platelets from the circulation is partly mediated by recognition of clustered beta-N-acetylglucosamine on platelet surface glycoproteins by the alphaMbeta2 hepatic lectin receptor. Capping the exposed beta-N-acetylglucosamine residues by enzymatic galactosylation restored the circulation of short-term chilled murine platelets, introducing a novel method that allows for cold storage of platelet. Recent studies have, however, shown that galactosylation is not sufficient to restore circulation of long-term refrigerated platelets. Additional data indicate that differential carbohydrate-mediated mechanisms may exist for clearance of short-term and long-term cold-stored platelets.Room temperature storage of platelet products increases the risk of transfusion-mediated sepsis and accelerates platelet deterioration, limiting platelet shelf life. Recent evidence suggests that glycoengineering of platelets might allow for their cold storage, significantly improving the quality of platelet products.

Glycosphingolipids (GSL) are glycosylated polar lipids in cell membranes essential for development of vertebrates as well as Drosophila. Mutants that impair enzymes involved in biosynthesis of GSL sugar chains provide a means to assess the functions of the sugar chains in vivo. The Drosophila glycosyltransferases Egghead and Brainiac are responsible for the 2nd and 3rd steps of GSL sugar chain elongation. Mutants lacking these enzymes are lethal and the nature of the defects that occur has suggested that GSL might impact on signaling by the Notch and EGFR pathways. Here we report on characterization of enzymes involved in the 4th and 5th steps of GSL sugar chain elongation in vitro and explore the biological consequences of removing the enzymes involved in step 4 in vivo. Two beta4-N-Acetylgalactosyltransferase enzymes can carry out step 4 (beta4GalNAcTA and beta4GalNAcTB), and while they may have overlapping activity, the mutants produce distinct phenotypes. The beta4GalNAcTA mutant displays behavioral defects, which are also observed in viable brainiac mutants, suggesting that proper locomotion and coordination primarily depend on GSL elongation. beta4GalNAcTB mutant animal shows ventralization of ovarian follicle cells, which is caused by defective EGFR signaling between the oocyte and the dorsal follicle cells to specify dorsal fate. GSL sequentially elongated by Egh, Brn and beta4GalNAcTB in the oocyte contribute to this signaling pathway. Despite the similar enzymatic activity, we provide evidence that the two enzymes are not functionally redundant in vivo, but direct distinct developmental functions of GSL.

Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glc{szligbeta}1Cer [rightwards arrow] Man{szligbeta}4Glc{szligbeta}1Cer [MacCer] [rightwards arrow] GlcNAc{szligbeta}3Man{szligbeta}4Glc{szligbeta}1Cer [At₃Cer] [rightwards arrow] GalNAc{szligbeta}4- GlcNAc{szligbeta}3Man{szligbeta}4Glc{szligbeta}1Cer [At₄Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Gal{szligbeta}3Man{szligbeta}4Glc{szligbeta}1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAc[alpha]4Gal{szligbeta}3Man{szligbeta}4- Glc{szligbeta}1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAc[alpha]4 and Gal{szligbeta}3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAc{szligbeta}4 in the arthro-series penta- and hexaglycosylceramide structures (At₅Cer and At₆Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAc[alpha]4 and Gal{szligbeta}3 residues in dipteran GSL biosynthesis and implies the existence of an insect Gal{szligbeta}3-T capable of using Man{szligbeta}4Glc{szligbeta}1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

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.

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like α2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic potential.

N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.

MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen made. We recently demonstrated that a monoclonal antibody against the immunodominant Tn-MUC1 (GalNAc-α-MUC1) antigen induced ADCC in breast cancer cell lines, suggesting the feasibility of targeting combined glycopeptide epitopes in future passive cancer immunotherapy.

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O ‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O ‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O ‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O ‐glycoproteome with almost 3000 glycosites in over 600 O ‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O ‐glycosylation. The finding of unique subsets of O ‐glycoproteins in each cell line provides evidence that the O ‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O ‐glycoproteome should facilitate the exploration of how site‐specific O ‐glycosylation regulates protein function.

Protein microarray is a highly sensitive tool for antibody detection in serum. Monitoring of patients' antibody titers to specific antigens is increasingly employed in the diagnosis of several conditions, ranging from infectious diseases, allergies, autoimmune diseases, and cancer. In this protocol we present a detailed method for enzymatic generation of disease-specific O-glycopeptides and how to monitor the antibody response to these in serum using microarray technology.

Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E. P., Takio, K., S⊘rensen, T., Bonding, N., and Clausen, H.(1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the GalNAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase preparations was identified. Analysis of GalNAc-transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple GalNAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.

Mucin-type glycosylation [α-N-acetyl-D-galactosamine (α-GalNAc)-O-Ser/Thr] on proteins is initiated biosynthetically by 16 homologous isoforms of GalNAc-Ts (uridine diphosphate-GalNAc:polypeptide N-acetylgalactosaminyltransferases). All the GalNAc-Ts consist of a catalytic domain and a lectin domain. Previous reports of GalNAc-T assays toward peptides and α-GalNAc glycopeptides showed that the lectin domain recognized the sugar on the substrates and affected the reaction; however, the details are not clear. Here, we report a new strategy to give insight on the sugar recognition ability and the function of the GalNAc-T3 lectin domain using chemically synthesized natural-type (α-GalNAc-O-Thr) and unnatural-type [β-GalNAc-O-Thr, α-Fuc-O-Thr and β-GlcNAc-O-Thr] MUC5AC glycopeptides. GalNAc-T3 is one of isoforms expressed in various organs, its substrate specificity extensively characterized and its anomalous expression has been identified in several types of cancer (e.g. pancreas and stomach). The glycopeptides used in this study were designed based on a preliminary peptide assay with a sequence derived from the MUC5AC tandem repeat. Through GalNAc-T3 and lectin-inactivated GalNAc-T3, competition assays between the glycopeptide substrates and product analyses (MALDI-TOF MS, RP-HPLC and ETD-MS/MS), we show that the lectin domain strictly recognized GalNAc on the substrate and this specificity controlled the glycosylation pathway.

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage beta(2) integrin binding to beta-N-acetylglucosamine (beta-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the beta-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both beta(2) integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold.

Cold storage of platelets for transfusion is desirable to extend platelet storage times and to prevent bacterial growth. However, the rapid clearance of cold-stored platelets prevents their use. A novel method for preventing the rapid clearance of cold-stored platelets has previously been developed in a murine model. Cold storage induces the clustering and recognition of exposed β-N-acetylglucosamine (βGlcNAc) on platelet surfaces. Glycosylation of βGlcNAc residues with uridine 5′-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0°C-stored murine platelets. Based on this finding, we developed a similar glycosylation process by adding UDP-galactose to human apheresis platelets. A phase 1 clinical trial was conducted transfusing radiolabeled autologous apheresis platelets stored for 48 hours at 4°C with or without pretreatment with UDP-galactose. In contrast to the murine study, galactosylation of human platelets did not prevent the accelerated platelet clearance routinely observed after 4°C storage. We next developed a murine model of platelet storage for 48 hours at 4°C and showed that UDP-galactose treatment of murine platelets also did not prevent their rapid clearance, in agreement with the human platelet study. We conclude that different mechanisms of clearance may exist for short- and long-term cold-stored platelets.

Glycosylation of viral envelope proteins is important for infectivity and interaction with host immunity, however, our current knowledge of the functions of glycosylation is largely limited to N-glycosylation because it is difficult to predict and identify site-specific O-glycosylation. Here, we present a novel proteome-wide discovery strategy for O-glycosylation sites on viral envelope proteins using herpes simplex virus type 1 (HSV-1) as a model. We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins. Two of the identified glycosites found in glycoprotein B were previously implicated in virus attachment to immune cells. We show that HSV-1 infection distorts the secretory pathway and that infected cells accumulate glycoproteins with truncated O-glycans, nonetheless retaining the ability to elongate most of the surface glycans. With the use of precise gene editing, we further demonstrate that elongated O-glycans are essential for HSV-1 in human HaCaT keratinocytes, where HSV-1 produced markedly lower viral titers in HaCaT with abrogated O-glycans compared to the isogenic counterpart with normal O-glycans. The roles of O-linked glycosylation for viral entry, formation, secretion, and immune recognition are poorly understood, and the O-glycoproteomics strategy presented here now opens for unbiased discovery on all enveloped viruses., Author Summary Information on site-specific O-glycosylation of viral envelope glycoproteins is generally very limited despite important functions. We present a powerful mass-spectrometry based strategy to globally identify O-glycosylation sites on viral envelope proteins of a given virus in the context of a productive infection. We successfully utilized the strategy to map O-linked glycosylation sites on the complex HSV-1 virus demonstrating that O-glycosylation is widely distributed on most envelope proteins. Moreover, we used genetically engineered keratinocytes lacking O-glycan elongation capacity to demonstrate that O-linked glycans are indeed important for HSV-1 biology as HSV-1 particles produced in these cells had significantly lower titers compared to wild-type keratinocytes. These tools enable wider discovery and detailed analysis of the role of site-specific O-glycosylation in virology.

To the editors: Wu et al.(1) reported cytotoxic T lymphocyte (CTL) recognition of LLGVGTFVV HLA-A*02 binding MUC4-derived peptide in healthy individuals, and MUC4 has been considered a target in ca...

Nat. Methods 8, 977–982 (2011); published online 9 October 2011; corrected after print 8 January 2015 In the version of this article initially published, the T3M4 cell line used for the experiments was mislabeled as Capan-1. The error has been corrected in the HTML and PDF versions of the article.

The neurogenic Drosophila genes brainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis of egghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:beta Man beta 1,3-N-acetylglucosaminyltransferase (beta 3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc beta 1-3Man beta 1-4Glc beta 1-1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:beta Glc beta 1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man beta 1-4Glc beta 1-1Cer. Egghead is unique among eukaryotic glycosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids.