Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 synthase deficiency variants.

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 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 underlying the neural phenotypes associated with GM3SD are unknown. 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 cells carrying either variant, 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. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in 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 erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

Duckweed pectic-arabinogalactan-proteins can crosslink through borate diester bonds.

Material containing pectin and arabinogalactan-protein (AGP) was released and purified from Spirodela alcohol insoluble residues. Results of carbohydrate analyses and two-dimensional NMR spectroscopy suggest that this material is composed of apiogalacturonan and rhamnogalacturonan-I covalently attached to AGPs. 11B NMR spectroscopy indicated that some of the glycoses in this complex exist as their boric acid monoesters. Borate diesters were formed when the pectic-AGPs were allowed to react at pH above 6.2 with the boron-depleted pectic-AGPs, suggesting that in vitro two pectic-AGP molecules can crosslink to one another through borate. Borate diesters also formed when the pectic-AGPs were incubated with monomeric rhamnogalacturonan-II in the presence of Pb2+ ion at pH 9.2. This data presents evidence of the first wall polymer after rhamnogalacturonan-II to crosslink through borate diesters. We suggest that the formation of these borate-crosslinks may help Spirodela respond to high-pH condition.

Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation.

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.

Mucin O-glycans are natural inhibitors of Candida albicans pathogenicity.

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.

Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate.

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.

Global mapping of glycosylation pathways in human-derived cells.

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.

High expression of tissue O-linked glycans is associated with a malignant phenotype of cholangiocarcinoma.

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.

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor.

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.

Ganglioside GM2: a potential biomarker for 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)ß1-4 glucose (Glc)ß1-1'-ceramide), Gb3 (Galα1-4Galß1-4Glcß1-1'-ceramide), Gb4/Lc4 (N-acetylgalactosamine (GalNAc)ß1-3Galα1-4Galß1-4Glcß1-1'-ceramide/Galß1-4 N-acetylglucosamine (GlcNAc)ß1-3Galß1-4Glcß1-1'-ceramide), GM3 (N-acetylneuraminic acid (NeuAc)2-3Galß1-4Glcß1-1'-ceramide), GM2 (GalNAcß1-4[NeuAc2-3]Galß1-4Glcß1-1'-ceramide), GM1 (Galß1-3GalNAcß1-4[NeuAc2-3]Galß1-4Glcß1-1'-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.

Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help.

The inherent molecular complexity of human pathogens requires that mammals evolved an adaptive immune system equipped to handle presentation of non-conventional MHC ligands derived from disease-causing agents, such as HIV-1 envelope (Env) glycoprotein. Here, we report that a CD4+ T cell repertoire recognizes a glycopeptide epitope on gp120 presented by MHCII pathway. This glycopeptide is strongly immunogenic in eliciting glycan-dependent cellular and humoral immune responses. The glycopeptide specific CD4+ T cells display a prominent feature of Th2 and Th17 differentiation and exert high efficacy and potency to help Env trimer humoral immune responses. Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells. Our identification of gp120 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV-1 infection.

Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions.

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.

Metabolic labeling of HIV-1 envelope glycoprotein gp120 to elucidate the effect of gp120 glycosylation on antigen uptake.

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.

Increased expression of the high-mannose M6N2 and NeuAc3H3N3M3N2F tri-antennary N-glycans in cholangiocarcinoma.

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.

O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

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.

Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis.

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 ß elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously.

Glycomic and glycoproteomic analysis of glycoproteins-a tutorial.

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.

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes.

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.

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.

An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.

Differential Expression of O-glycoprotein Glycans in Cholangiocarcinoma Cell Lines.

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.

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

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.