Core-Fucosylated Tetra-Antennary N-Glycan Containing A Single N-Acetyllactosamine Branch Is Associated with Poor Survival Outcome in Breast Cancer.

(1) Glycoproteins account for ~80% of proteins located at the cell surface and in the extracellular matrix. A growing body of evidence indicates that α-L-fucose protein modifications contribute to breast cancer progression and metastatic disease. (2) Using a combination of techniques, including matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) based in cell and on tissue imaging and glycan sequencing using exoglycosidase analysis coupled to hydrophilic interaction ultra-high performance liquid chromatography (HILIC UPLC), we establish that a core-fucosylated tetra-antennary glycan containing a single N-acetyllactosamine (F(6)A4G4Lac1) is associated with poor clinical outcomes in breast cancer, including lymph node metastasis, recurrent disease, and reduced survival. (3) This study is the first to identify a single N-glycan, F(6)A4G4Lac1, as having a correlation with poor clinical outcomes in breast cancer.

Moving beyond the van Krevelen Diagram: A New Stoichiometric Approach for Compound Classification in Organisms.

van Krevelen diagrams (O/C vs H/C ratios of elemental formulas) have been widely used in studies to obtain an estimation of the main compound categories present in environmental samples. However, the limits defining a specific compound category based solely on O/C and H/C ratios of elemental formulas have never been accurately listed or proposed to classify metabolites in biological samples. Furthermore, while O/C vs H/C ratios of elemental formulas can provide an overview of the compound categories, such classification is inefficient because of the large overlap among different compound categories along both axes. We propose a more accurate compound classification for biological samples analyzed by high-resolution mass spectrometry based on an assessment of the C/H/O/N/P stoichiometric ratios of over 130 000 elemental formulas of compounds classified in 6 main categories: lipids, peptides, amino sugars, carbohydrates, nucleotides, and phytochemical compounds (oxy-aromatic compounds). Our multidimensional stoichiometric compound classification (MSCC) constraints showed a highly accurate categorization of elemental formulas to the main compound categories in biological samples with over 98% of accuracy representing a substantial improvement over any classification based on the classic van Krevelen diagram. This method represents a signficant step forward in environmental research, especially ecological stoichiometry and eco-metabolomics studies, by providing a novel and robust tool to improve our understanding of the ecosystem structure and function through the chemical characterization of biological samples.

slan-defined subsets of CD16-positive monocytes: impact of granulomatous inflammation and M-CSF receptor mutation.

Human monocytes are subdivided into classical, intermediate, and nonclassical subsets, but there is no unequivocal strategy to dissect the latter 2 cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14(+)CD16(++) nonclassical monocytes and slan-negative CD14(++)CD16(+) intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of major histocompatibility complex class II genes, whereas a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering, the slan-positive nonclassical monocytes cluster with monocytes and are clearly distinct from CD1c(+) dendritic cells. In clinical studies, we show a selective increase of the slan-negative intermediate monocytes to >100 cells per microliter in patients with sarcoidosis and a fivefold depletion of the slan-positive monocytes in patients with hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), which is caused by macrophage colony-stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-based definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings.

N-glycosylation profiling of porcine reproductive and respiratory syndrome virus envelope glycoprotein 5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV.

CHoMP: a chemoenzymatic histology method using clickable probes.

The characterization of aberrant glycosylation patterns in biopsied patient samples represents a remarkable challenge for scientists and medical doctors due to the lack of specific methods for detection. Here, we report the development of a histological method, dubbed CHoMP-chemoenzymatic histology of membrane polysaccharides-for analyzing glycosylation patterns in mammalian tissues. This method exploits a recombinant glycosyltransferase to transfer a monosaccharide analogue equipped with a chemical handle to a specific cell-surface glycan target, which can then be derivatized with imaging probes by using bioorthogonal click chemistry for visualization. We applied CHoMP to survey changes in expression of N-acetyllactosamine (LacNAc) in human samples from patients afflicted with lung adenocarcinoma and observed a sharp decrease in expression levels between normal and early grade tumors, thus suggesting a potential application of this technique in early cancer diagnosis.

Impact of chemotherapeutic agents on the immunostimulatory properties of human 6-sulfo LacNAc+ (slan) dendritic cells.

Chemotherapy is an important treatment modality for many patients with advanced cancer. Recent data revealed that certain chemotherapeutic agents differentially affect maturation, cytokine production and T-cell stimulatory capacity of dendritic cells (DCs), which play a crucial role in the induction of antitumor immunity. Whereas most reports are based on mouse or human monocyte-derived DCs, studies investigating the direct effect of chemotherapeutic drugs on native human DCs are rather limited. Here, we evaluated the impact of various chemotherapeutic drugs on the immunostimulatory properties of 6-sulfo LacNAc(+) (slan) DCs, representing a major subpopulation of human blood DCs. Because of their various antitumor effects, slanDCs may essentially contribute to the immune defense against tumors. We demonstrated that doxorubicin and vinblastine significantly impair the release of tumor necrosis factor-α, interleukin (IL)-6 and IL-12 by slanDCs. Functional data revealed that both drugs inhibit slanDC-mediated proliferation of T lymphocytes and their capacity to differentiate naive CD4(+) T cells into proinflammatory T-helper type I cells. Furthermore, these agents markedly suppressed the ability of slanDCs to stimulate interferon-γ secretion by natural killer (NK) cells. In contrast, paclitaxel, mitomycin C and methotrexate sustained the ability of slanDCs to produce proinflammatory cytokines and their potential to activate T-lymphocytes and NK cells. These results indicate that doxorubicin and vinblastine impair the ability of native human DCs to stimulate important immune effector cells, whereas methotrexate, mitomycin C and paclitaxel maintain their immunostimulatory properties. These novel findings may have implications for the design of treatment modalities for tumor patients combining immunotherapeutic strategies and chemotherapy.

The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern.

Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells presented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.

Glycomic and proteomic profiling of pancreatic cyst fluids identifies hyperfucosylated lactosamines on the N-linked glycans of overexpressed glycoproteins.

Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on ß-lactosamine extensions. Following the observation of these "hyperfucosylated" glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively, following A. aurantia lectin enrichment.

Extracorporeal photopheresis efficiently impairs the proinflammatory capacity of human 6-sulfo LacNAc dendritic cells.

BACKGROUND: Extracorporeal photopheresis (ECP) emerged as a promising treatment modality for steroid-refractory graft-versus-host disease (GVHD), which represents a major complication of allogeneic hematopoietic stem-cell transplantation. Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses and play a crucial role in the initiation and maintainance of GVHD. This study evaluated the direct impact of ECP on the proinflammatory capacity of 6-sulfo LacNAc (slan) DCs, representing a major subpopulation of human blood DCs. METHODS: SlanDCs were isolated from ECP-treated or untreated blood of healthy donors or GVHD patients by immunomagnetic isolation. The maturation of slanDC was determined by flow cytometry. Cytokine production of slanDCs was measured by enzyme-linked immunosorbent assay. SlanDC-mediated T-cell proliferation was evaluated by H-thymidine incorporation. SlanDC-mediated T-cell programming was determined by flow cytometry. RESULTS: ECP efficiently impairs the spontaneous maturation and secretion of proinflammatory tumor necrosis factor-alpha, interleukin-1beta, and interleukin-12 by slanDCs. Furthermore, ECP markedly inhibits slanDC-induced proliferation of CD4 and CD8 T cells and polarization of naïve CD4 T lymphocytes into Th1 cells. CONCLUSIONS: These novel findings indicate that ECP efficiently impairs the proinflammatory capacity of slanDCs, which may represent an important mechanism for the therapeutic efficiency of ECP in GVHD.

Critical assessment of the applicability of gas chromatography-combustion-isotope ratio mass spectrometry to determine amino sugar dynamics in soil.

Amino sugars in soils have been used as markers of microbial necromass and to determine the relative contribution of bacterial and fungal residues to soil organic matter. However, little is known about the dynamics of amino sugars in soil. This is partly because of a lack of adequate techniques to determine 'turnover rates' of amino sugars in soil. We conducted an incubation experiment where (13)C-labeled organic substrates of different quality were added to a sandy soil. The objectives were to evaluate the applicability of compound-specific stable isotope analysis via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the determination of (13)C amino sugars and to demonstrate amino sugar dynamics in soil. We found total analytical errors between 0.8 and 2.6 per thousand for the delta(13)C-values of the soil amino sugars as a result of the required delta(13)C-corrections for isotopic alterations due to derivatization, isotopic fractionation and analytical conditions. Furthermore, the delta(13)C-values of internal standards in samples determined via GC-C-IRMS deviated considerably from the delta(13)C-values of the pure compounds determined via elemental analyzer IRMS (with a variation of 9 to 10 per thousand between the first and third quartile among all samples). This questions the applicability of GC-C-IRMS for soil amino sugar analysis. Liquid chromatography-combustion-IRMS (LC-C-IRMS) might be a promising alternative since derivatization, one of the main sources of error when using GC-C-IRMS, is eliminated from the procedure. The high (13)C-enrichment of the substrate allowed for the detection of very high (13)C-labels in soil amino sugars after 1 week of incubation, while no significant differences in amino sugar concentrations over time and across treatments were observed. This suggests steady-state conditions upon substrate addition, i.e. amino sugar formation equalled amino sugar decomposition. Furthermore, higher quality substrates seemed to favor the production of fungal-derived amino sugars.

Interleukin-1beta induces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene.

We previously demonstrated that human hepatocellular carcinoma-derived HuH-7 cells stimulated with interleukin-1beta (IL-1beta) produce alpha(1)-acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH-7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL-1 beta compared with control, as indicated by anti-sLeX antibody binding. Furthermore, expression of 2,3-sialylated N-acetyllactosamine increased gradually up to 48 h after IL-1 beta stimulation; this preceded the increase in sLeX expression. Increases in alpha 2,3-sialyltransferase activity also preceded increases in alpha1,3-fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH-7 cells stimulated with IL- 1beta were increased at 2-4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL-1 beta-induced sLeX expression on HuH-7 cells was suppressed by transfection of gene-specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL-1 beta induces expression of sLeX on HuH-7 cells by enhanced expression of FUT VI and ST3Gal IV gene.

A gas chromatographic/mass spectrometric method for tracing the microbial conversion of glucose into amino sugars in soil.

Amino sugars in soils are heterogeneous and have been used as microbial residue biomarkers to investigate the microbial contribution to soil organic matter. However, it is not clear what the available carbon source is and how glucose is utilized for the synthesis of soil amino sugars. This paper presents a new gas chromatography/mass spectrometry (GC/MS) approach for the identification of 13C incorporation into three amino sugars, D-glucosamine, D-galactosamine, and muramic acid, in soil incubated with U-13C-glucose. Method evaluation showed that the chemical ionization (CI) mode was suitable for all these amino sugars, but that electron impact (EI) mode was applicable only to glucosamine and galactosamine. The 13C conversion rate was estimated based on the abundance ratio of the ions corresponding to the masses of the ions F+n and F (where n is the skeleton carbon number in the fragment ions F of the amino sugars) and calculated as atom percentage excess. The reproducibility of the method was excellent and clearly adequate for the present purpose. In addition, the new approach is highly accurate as tested with mixtures of U-13C-glucose and natural glucose.

MUC5B glycosylation in human saliva reflects blood group and secretor status.

This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from individuals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive beta-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O individuals into larger population (29 individuals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 individuals. The remaining six individuals were characterized by a high degree of sialylation. These individuals were assigned as nonsecretors, whereas blood group-expressing individuals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Le(a)) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between individuals and that some of this variation is owing to blood group and secretor status.

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases.

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more heavily N-glycosylated than G(N). No evidence was observed for the presence of O-linked oligosaccharides on G(N) or G(C).

Evidence for early infection of nonneoplastic natural killer cells by Epstein-Barr virus.

We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.

Evaluation of amino sugar, low molecular peptide and amino acid impurities of biotechnologically produced amino acids by means of CE.

As the second part of our studies on the impurity profiles of amino acids produced by biotechnological processes, a micellar electrokinetic chromatography (MEKC) was applied to determine amino sugars, low molecular peptides and amino acids as potential expected impurities at a level 0.1% w/w. 3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) was found to be suitable as a labeling reagent for laser induced fluorescence (LIF) detection. The labeling reaction was optimized to achieve maximum reaction yields and reproducibility for all groups of substances. The 'level 0.1%' was chosen as quantities/concentrations in such a way that the major reagent peak becomes comparable with the impurities at this level, and the minor reagent peaks (<0.01%) are not able to cover other peaks at the level of interest. The reaction optimization and the validation studies were performed with model mixtures of representative amino sugars, low molecular peptides and amino acids. A linearity range of labeling of three orders of magnitude was achieved. The total precision of the method, including the labeling reproducibility was studied and for all test substances relative standard deviation less than 5% were obtained. The accuracy was evaluated by performing recovery experiments at three concentrations covered a 50-200% interval of the level 0.1%. Confidence intervals below +/-2.5% (lambda=0.05, n=9) of the target were found sufficient for purity tests.

Comparison of oligosaccharides derived from salivary mucin of Japanese secretor and non-secretor individuals of blood group type-A.

Comparison of oligosaccharide components derived from salivary mucin was performed between secretor and non-secretor individuals. Salivary mucin was collected from four secretors and three non-secretors having blood group type-A. Compositional analysis showed that the contents of galactose and N-acetylglucosamine in the non-secretor were higher than those in the secretor. The O-linked oligosaccharides obtained by treatment with alkaline borohydride were separated by gel filtration using Sephadex G-50. The results indicated that the size of the type-A active oligosaccharides from the secretor was similar to or smaller than that of the non-secretor. Ion-exchange chromatography showed that the secretors had strong type-A activities in both the neutral and acidic fractions but the non-secretors showed type-A activity mainly in the neutral fraction. These results suggest that compositional differences in blood group substances exist between secretors and non-secretors.