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BACKGROUND: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. OBJECTIVES: The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. METHODS: GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans. RESULTS: We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain. CONCLUSIONS: This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.

BackgroundPlatelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand Factor (VWF). GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, VWF binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized.ObjectivesThe aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures.MethodsGPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry (MS) glycomics, and MS glycoproteomics to define glycosites and the structures of the attached glycans.ResultsWe identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABH blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified at least 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain (MSD).ConclusionsThis comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.Essentials-Glycosylation of glycoprotein Ibα (GPIbα) is important for platelet function.-We report a comprehensive and site-specific analysis of human GPIbα glycosylation.-GPIbα carries sialoglycans, Tn antigen, T antigen, and ABO blood group (ABH) antigens.-We experimentally determined 48 O-glycosites and 1 N-glycosite by mass spectrometry.

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a single α3-linked sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms that account for neural phenotypes associated with GM3SD are not known. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in iPSCs and NCCs from both variants, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated, GSLs compared to WT. Shifts in ceramide profiles associated with iPSC and NCC GSLs were also detected in GM3SD variants. Altered GSL profiles in the GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotnib, an inhibitor of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase increased protein O-GlcNAcylation and significantly rescued baseline and erlotnib-induced apoptosis. Collectively, these findings indicate broad effects on cell signaling during differentiation of GM3SD patient-derived iPSCs to NCCs. Thus, human GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling, cell survival, and neural differentiation.

Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

Objective This study aimed to investigate the expression of O-linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue. Methods Sixty patients with cholangiocarcinoma were included in the study. Permethylated O-linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1. Results O-linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight O-linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II–IV), lymphatic invasion, and vascular invasion. Conclusion Our study shows expression of O-linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.

Objective To investigate the expression of glycosphingolipids in serum and tissue from patients with cholangiocarcinoma compared with healthy controls. Methods Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used to demonstrate the comparative structural glycomics of glycosphingolipids in serum from patients with cholangiocarcinoma (n=15), compared with healthy controls (n = 15). GM2 expression in cholangiocarcinoma tissues (n = 60) was evaluated by immunohistochemistry. Results Eleven glycosphingolipids were detected by NSI-MSn: CMH (ceramide monohexose), Lac-Cer (galactose (Gal)β1-4 glucose (Glc)β1-1'-ceramide), Gb3 (Galα1-4Galβ1-4Glcβ1-1'-ceramide), Gb4/Lc4 (N-acetylgalactosamine (GalNAc)β1-3Galα1-4Galβ1-4Glcβ1-1'-ceramide/Galβ1-4 N-acetylglucosamine (GlcNAc)β1-3Galβ1-4Glcβ1-1'-ceramide), GM3 (N-acetylneuraminic acid (NeuAc)2-3Galβ1-4Glcβ1-1'-ceramide), GM2 (GalNAcβ1-4[NeuAc2-3]Galβ1-4Glcβ1-1'-ceramide), GM1 (Galβ1-3GalNAcβ1-4[NeuAc2-3]Galβ1-4Glcβ1-1'-ceramide), hFA (hydroxylated fatty acid)-CMH, hFA-Lac-Cer, hFA-Gb3, and hFA-GM3. Lac-Cer was the most abundant structure among the lactosides and globosides (normal, 24.40% ± 0.11%; tumor, 24.61% ± 2.10%), while GM3 predominated among the gangliosides (normal, 29.14% ± 1.31%; tumor, 30.53% ± 4.04%). The two glycosphingolipids that significantly differed between healthy controls and patients with cholangiocarcinoma were Gb3 and GM2. High expression of GM2 was associated with vascular invasion in tissue from patients with cholangiocarcinoma. Conclusions Altered expression of glycosphingolipids in tissue and serum from patients with cholangiocarcinoma may contribute to tumor growth and progression. The ganglioside GM2, which significantly increased in the serum of patients with cholangiocarcinoma, represents a promising target as a biomarker for cholangiocarcinoma.

HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations., Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components. IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor’s ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR’s extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity., Olson et al. report the crystal structures of the N-terminal five domains of human cation-independent mannose 6-phosphate receptor (CI-MPR) suggesting a binding- and pH-induced conformational change. Their data may explain how high-affinity carbohydrate-binding is achieved through allosteric domain cooperativity.

The green colonial microalga Botryococcus braunii produces large amounts of liquid hydrocarbons that can be converted into transportation fuels. Colony cells are held together by a complex extracellular matrix (ECM) made up of a cross-linked long-chain hydrocarbon network around which liquid hydrocarbons are stored, a retaining wall for holding hydrocarbons within the cross-linked hydrocarbon network, and a polysaccharide fibrillar sheath radiating from the retaining wall and surrounding the entire colony. Analysis of “shells” shed from cell apical regions during cell division and containing the retaining wall and polysaccharide fibers shows association of a single protein where the fibers meet the retaining wall, suggesting involvement of this protein in polysaccharide fiber formation. Here we use peptide mass fingerprinting and bioinformatics to identify this protein called p oly s accharide a ssociated p rotein (PSAP). PSAP does not show similarity to any protein in databases, but contains several Proline-rich domains. Staining studies confirm PSAP as a glycoprotein, and mass spectrometry analysis identified ten N -linked glycosylation sites comprising seven different glycans containing mainly mannose and N -acetylglucosamine. Three of these glycans also contain fucose, with one of these glycans being unusual since it also contains arabinose. Additionally, four hydroxyproline residues have short O -linked glycans of mainly arabinose and galactose, with one also containing a 6-deoxyhexose. PSAP secretion and localization to shell material is confirmed using western blot analysis and microscopy. These studies indicate PSAP contains unique glycans and suggest its involvement in ECM polysaccharide fiber biosynthesis.

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box-binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box-binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box-combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells.

The green microalga Botryococcus braunii forms a colony of cells held together by an intricate extracellular matrix. This extracellular matrix also acts as a storage location for liquid hydrocarbons initially produced inside the cells. These hydrocarbons can be used as feedstocks to produce transportation fuels. In the B race of B. braunii, the extracellular matrix is composed of several elements: a cross-linked hydrocarbon network filled with the liquid hydrocarbons, a retaining wall to hold the hydrocarbons in the extracellular matrix, an arabinogalactan polysaccharide fibrillar sheath that extends outward from the retaining wall, and a single protein known as polysaccharide associated protein is found at the base of the polysaccharide fibers where they connect to the retaining wall. During growth, colonies of the B race of B. braunii shed fragments of the retaining wall with polysaccharide fibrils attached into the media. Here we describe the isolation and structural analysis of these polysaccharides using a series of chemical degradations in combination with glycosyl residue composition and linkage analysis, as well as 2-dimensional nuclear magnetic resonance spectroscopy. The results demonstrate that the polysaccharide is highly branched, consisting of a galactan backbone with short branches comprised of d -galactopyranose, l -arabinofuranose, 4-O-methyl- d -glucopyranuronic acid, and 6-deoxy- d -altropyranose. To our knowledge, this is the first description of 6-deoxyaltrose in algae or plants.

To overcome the challenges in the analysis of protein glycosylation, we have developed a comprehensive and universal tool through permethylation of glycopeptides and their tandem mass spectrometric analysis. This method has the potential to simplify glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. Moreover, glycans with unique glycosidic linkages, particularly from prokaryotes, which are resistant to enzymatic or chemical release, could also be detected and analyzed by this methodology. Here we present a strategy for the permethylation of intact glycopeptides, obtained via controlled protease digest, and their characterization by using advanced mass spectrometry. We used bovine RNase B, human transferrin, and bovine fetuin as models to demonstrate the feasibility of the method. Remarkably, the glycan patterns, glycosylation site, and their occupancy by N-glycans are all detected and identified in a single experimental procedure. Acquisition on a high resolution tandem-MSn system with fragmentation methodologies such as high-energy collision dissociation (HCD) and collision induced dissociation (CID), provided the complete sequence of the glycan structures attached to the peptides. The behavior of 20 natural amino acids under the basic permethylation conditions was probed by permethylating a library of short synthetic peptides. Our studies indicate that the permethylation imparts simple, limited, and predictable chemical transformations on peptides and do not interfere with the interpretation of MS/MS data. In addition to this, permethylated O-glycans in unreduced form (released by β elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously.

The structural analysis of glycoproteins is a challenging endeavor and is under steadily increasing demand, but only a very limited number of labs have the expertise required to accomplish this task. This tutorial is aimed at researchers from the fields of molecular biology and biochemistry that have discovered that glycoproteins are important in their biological research and are looking for the tools to elucidate their structure. It provides brief descriptions of the major and most common analytical techniques used in glycomics and glycoproteomics analysis, including explanations of the rationales for individual steps and references to published literature containing the experimental details necessary to carry out the analyses. Glycomics includes the comprehensive study of the structure and function of the glycans expressed in a given cell or organism along with identification of all the genes that encode glycoproteins and glycosyltransferases. Glycoproteomics which is subset of both glycomics and proteomics is the identification and characterization of proteins bearing carbohydrates as posttranslational modification. This tutorial is designed to ease entry into the glycomics and glycoproteomics field for those without prior carbohydrate analysis experience.

Glycans are one of the fundamental classes of macromolecules and are involved in a broad range of biological phenomena. A large variety of glycan structures can be synthesized depending on tissue or cell types and environmental changes. Here, we developed a comprehensive glycosylation mapping tool, termed GlycoMaple, to visualize and estimate glycan structures based on gene expression. We informatically selected 950 genes involved in glycosylation and its regulation. Expression profiles of these genes were mapped onto global glycan metabolic pathways to predict glycan structures, which were confirmed using glycomic analyses. Based on the predictions of N-glycan processing, we constructed 40 knockout HEK293 cell lines and analyzed the effects of gene knockout on glycan structures. Finally, the glycan structures of 64 cell lines, 37 tissues, and primary colon tumor tissues were estimated and compared using publicly available databases. Our systematic approach can accelerate glycan analyses and engineering in mammalian cells.

Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by NeuAc2Gal1GalNAc1. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA.

The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking. Here, we integrate metabolic labeling and click chemistry technology with recombinant gp120 expression to demonstrate that gp120 glycans could be specifically labeled and directly detected. Selective labeling of gp120 by N-azidoacetylmannosamine (ManNAz) and N-azidoacetylgalactosamine (GalNAz) incorporation into the gp120 glycan shield was characterized by MS of tryptic glycopeptides. By using metabolically labeled gp120, we investigated the impact of gp120 glycosylation on its interaction with host cells and demonstrated that oligomannose enrichment and sialic acid deficiency drastically enhanced gp120 uptake by bone marrow-derived dendritic cells. Collectively, our data reveal an effective labeling and detection method for gp120, serving as a tool for functional characterization of the gp120 glycans and potentially other glycosylated proteins.

Changes in protein glycosylation have been reported in various types of cancer, including cholangiocarcinoma (CCA). Nanospray ionization-linear ion trap mass spectrometry (NSI-MS n ) was used in the present study to determine the comparative structural glycomics of the N-linked glycans in the serum of patients with CCA compared with healthy controls. A total of 5 high-mannose and 4 complex N-linked glycans were detected. Mannose7-N-acetyl-glucosamine2 was the most abundant structure among the high-mannose types (control 12.12±2.54 vs. CCA 9.27±2.66%), whereas NeuAc2H2N2M3N2 predominated the complex types (control 61.17±2.55 vs. CCA 64.68±4.23%). The expression of 3 different N-glycans differed significantly between the CCA cases and controls. These included mannose6-N-acetyl-glucosamine2 (P=0.044), mannose9-N-acetyl-glucosamine2 (Ρ=0.030) and NeuAc3H3N3M3N2F (Ρ=0.002). These three glycan structures may therefore be associated with tumor progression in CCA and may be useful for its diagnosis.

ABO blood groups have long been associated with cardiovascular disease, thrombosis, and acute coronary syndromes. Many studies over the years have shown type O blood group to be associated with lower risk of cardiovascular disease than non–type O blood groups. However, the mechanisms underlying this association remain unclear. Although ABO blood group is associated with variations in concentrations of circulating von Willebrand Factor and other endothelial cell adhesion molecules, ABO antigens are also present on several platelet surface glycoproteins and glycosphingolipids. As we highlight in this platelet-centric review, these glycomic modifications may affect platelet function in arterial thrombosis. More broadly, improving our understanding of the role of platelet glycan modifications in acute coronary syndromes may inform future diagnostics and therapeutics for cardiovascular diseases.

Glycogen is a branched polymer of glucose that acts as an energy reserve in many cell types. Glycogen contains trace amounts of covalent phosphate, in the range of 1 phosphate per 500-2000 glucose residues depending on the source. The function, if any, is unknown, but in at least one genetic disease, the progressive myoclonic epilepsy Lafora disease, excessive phosphorylation of glycogen has been implicated in the pathology by disturbing glycogen structure. Some 90% of Lafora cases are attributed to mutations of the EPM2A or EPM2B genes, and mice with either gene disrupted accumulate hyperphosphorylated glycogen. It is, therefore, of importance to understand the chemistry of glycogen phosphorylation. Rabbit skeletal muscle glycogen contained covalent phosphate as monoesters of C2, C3, and C6 carbons of glucose residues based on analyses of phospho-oligosaccharides by NMR. Furthermore, using a sensitive assay for glucose 6-P in hydrolysates of glycogen coupled with measurement of total phosphate, we determined the proportion of C6 phosphorylation in rabbit muscle glycogen to be ∼20%. C6 phosphorylation also accounted for ∼20% of the covalent phosphate in wild type mouse muscle glycogen. Glycogen phosphorylation in Epm2a(-/-) and Epm2b(-/-) mice was increased 8- and 4-fold compared with wild type mice, but the proportion of C6 phosphorylation remained unchanged at ∼20%. Therefore, our results suggest that C2, C3, and/or C6 phosphate could all contribute to abnormal glycogen structure or to Lafora disease.

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box–binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box–binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box–binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box–combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells.

Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by IsoTaG were found to be comparable to released N-glycans identified by permethylation analysis. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.

Aims Tea (green, oolong, and black) is the second most widely consumed beverage worldwide, second only to water. Aside from a few reported adverse effects, tea, particularly green tea, appears to be beneficial for human health. In the case described herein, a male experienced several transient ischemic attack-like symptoms immediately following the consumption of a cup of high quality oolong tea. A thorough medical evaluation uncovered no evidence of such an attack and leads to the suggestion of a heretofore unreported response to oolong tea. Presentation of case A 72-year old male with hypertension and atrial fibrillation, who takes valsartan/hydrochlorothiazide to control hypertension and warfarin to reduce the risk of thrombosis and thromboembolism, presented at the emergency room of a local hospital describing several transient ischemic attack-like symptoms immediately after consuming a cup of oolong tea. His symptoms included presyncope, disequilibrium, bilateral hand parathesias, mild dysphasia, and visual problems (but apparently not presbyopia or amaurosis fugax), all of which had disappeared in approximately two hours after drinking the tea. (Mild presyncope was previously noted by the patient when ingesting a strong green tea.) No unusual features emerged from his physical examination, and his blood work was unremarkable except for elevation of his partial thromboplastin time (39 sec) and prothrombin time (22.5 sec), giving an international reference of 2.0, all consistent with the effects of warfarin. A battery of tests by the emergency room physician, a cardiologist, and a neurologist, e.g. electrocardiogram, brain computerized tomography, 2-dimensional transthoracic echocardiogram, brain magnetic resonance imaging, with and without 20 ml Gadolinium, and a magnetic resonance angiogram, confirmed the earlier diagnosis of atrial fibrillation but disclosed no additional malfunction in his heart. His brain showed no evidence of a prior hemorrhage, and his carotid arteries were clear. Methodology and results Analysis of the oolong tea by high performance liquid chromatography and mass spectrometry identified the major catechins and two methylxanthines, caffeine and theophylline, as well as other constituents, but there was no evidence of any extraneous chemicals that could lead to the symptoms. Conclusion In view of the rapid onset of symptoms after the consumption of oolong tea, bilateral as opposed to unilateral parathesis, and the absence of any evidence of a hemorrhage or the presence of impurities in the tea, we suggest that the transient ischemic attack-like symptoms could possibly be attributable to one or more components of the oolong tea and was not an atypical magnetic resonance imaging-negative transient ischemic attack.

Changes in protein glycosylation have been reported in various types of cancer, including cholangiocarcinoma (CCA). Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used in the present study to determine the comparative structural glycomics of the N-linked glycans in the serum of patients with CCA compared with healthy controls. A total of 5 high-mannose and 4 complex N-linked glycans were detected. Mannose7-N-acetyl-glucosamine2 was the most abundant structure among the high-mannose types (control 12.12±2.54 vs. CCA 9.27±2.66%), whereas NeuAc2H2N2M3N2 predominated the complex types (control 61.17±2.55 vs. CCA 64.68±4.23%). The expression of 3 different N-glycans differed significantly between the CCA cases and controls. These included mannose6-N-acetyl-glucosamine2 (P=0.044), mannose9-N-acetyl-glucosamine2 (Ρ=0.030) and NeuAc3H3N3M3N2F (Ρ=0.002). These three glycan structures may therefore be associated with tumor progression in CCA and may be useful for its diagnosis.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses., Pneumocystis jirovecii is a fungus that can cause life-threatening pneumonia in immunocompromised patients. Here, the authors sequence the genomes of P. jirovecii and two other Pneumocystis species, and show the unexpected absence of chitin (a near universal fungal cell wall component).

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multiinstitutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/ Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful. Molecular & Cellular Proteomics 9: 719-727, 2010.

Mindin (spondin-2) is an extracellular matrix protein of unknown structure that is required for efficient T-cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N-terminal F-spondin (FS) domain and C-terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8-A resolution. The structure revealed an eight-stranded antiparallel beta-sandwich motif resembling that of membrane-targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C-mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

Cardiovascular diseases (CVD) are the most common causes of death in developed countries and their prevalence rate is rapidly growing. Understanding of changes in proteins, sphingolipids and their ...

Cardiovascular diseases are the most common causes of death in developed countries and their prevalence rate is rapidly growing in developing countries. Genome-wide association studies (GWAS) have identified associations between ABO blood group status and HLBS (heart, lung, blood, and sleep) disorders. ABO glycans are expressed on glycoproteins and glycosphingolipids (GSLs) of cells and tissues relevant to HLBS disorders,including endothelial cells and platelets. Several major HLBS disorders, particularly acute myocardial infarction and acute lung injury, are characterized by endothelium dysfunction and platelet-driven thrombosis. Therefore, a comprehensive understanding of the disposition of ABO glycan expression by endothelial cells and platelets is essential for elucidating the mechanisms by which these glycans influence risk for cardiovascular disease. Toward this end, we have undertaken the structural characterization of GSL, and N- and O-linked glycoprotein glycans in resting and activated platelets...

Genome-wide association studies have suggested that, compared to non-O blood types, the ABO O blood type phenotype and related genotypes are associated with protection from CVD. To test the hypothesis that ABO glycosyltransferase modulates endothelial functional pathways and glycan profile, we differentiated adipose stem cell (ASC)-derived induced pluripotent stem cell (iPSC) from individuals of different ABO blood types to endothelial cells (ECs). iPSC-ECs exhibited cobble-stone morphology, demonstrated capillary tube formation on matrigel, and incorporated acetylated low-density lipoprotein. FACS analysis indicates expression of both CD31 and vascular endothelial growth factor receptor-2 (VEGFR2). Immunocytochemical analysis of iPSC-ECs revealed positive staining for EC markers e.g., CD31, vascular endothelial cadherin (CD144), and von Willebrand factor (vWF). The phenotypic characteristics of iPSC-ECs were similar to that of human coronary arterial endothelial cells (hCAECs). The adhesion pattern of iP...

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPOP) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM (21%). The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production. Published version

International audience; Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.

SummaryGlycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure, and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

Cellulosic biomass is an abundant and promising energy source. To make cellulosic biofuels competitive against conventional fuels, conversion of rigid plant materials into sugars must become efficient and cost-effective. During cellulose degradation, cellulolytic enzymes generate cellobiose (β-(1→4)-glucose dimer) molecules, which in turn inhibit such enzymes by negative feedback. β-Glucosidases (BGLs) cleave cellobiose into glucose monomers, assisting overall cellulolytic activities. Therefore, BGLs are essential for efficient conversion of cellulosic biomass into biofuels, and it is important to characterize newly isolated BGLs for useful traits. Here, we report our discovery that the indigenous Taiwanese fungus Chaetomella raphigera strain D2 produces two molecular weight variants of a single BGL, D2-BGL (shortened to "D2"), which differ in O-glycosylation. The more extensively O-glycosylated form of native D2 (nD2L) has increased activity toward the natural substrate, cellobiose, compared to the less O-glycosylated form (nD2S). nD2L is more stable at 60°C, in acidic pH, and in the presence of the ionic detergent sodium dodecyl sulfate than nD2S. Furthermore, unlike nD2S, nD2L does not display substrate inhibition by an artificial substrate p-nitrophenyl glucopyranoside (pNPG), and the glucose feedback inhibition kinetics of nD2L is competitive (while it is non-competitive for nD2S), suggesting that these two glycovariants of D2 bind substrates differently. Interestingly, D2 produced in a heterologous system, Pichia pastoris, closely mimics properties of nD2S. Our studies suggest that O-glycosylation of D2 is important in determining its catalytic and biochemical properties.