Biomedical subjects

Anne Dell

Publications and source records attributed to Anne Dell.

At least 19 recordsLinked to original sources

PubMed2007

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

Carbohydrate Conformation↗

PubMed2006

Inactivation of Corynebacterium glutamicum NCgl0452 and the role of MgtA in the biosynthesis of a novel mannosylated glycolipid involved in lipomannan biosynthesis.

Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac(1)PIM(1)) to generate Ac(1)PIM(2). Herein, we describe the disruption of its probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type Corynebacterium glutamicum and the C. glutamicum::pimB mutant. Following a careful analysis, two related glycolipids, Gl-A and Gl-X, were found in the parent strain, but Gl-X was absent from the mutant. The biosynthesis of Gl-X was restored in the mutant by complementation with either Cg-pimB or Mt-pimB. Subsequent chemical analyses established Gl-X as 1,2-di-O-C(16)/C(18:1)-(alpha-d-mannopyranosyl)-(1-->4)-(alpha-d-glucopyranosyluronic acid)-(1-->3)-glycerol (ManGlcAGroAc(2)) and Gl-A as the precursor, GlcAGroAc(2). In addition, C. glutamicum::pimB was still able to produce Ac(1)PIM(2), suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc(2) from GlcAGroAc(2). Isolation of lipoglycans from C. glutamicum led to the identification of two related lipoglycans. The larger lipoglycan possessed a lipoarabinomannan-like structure, whereas the smaller lipoglycan was similar to lipomannan (LM). The absence of ManGlcA-GroAc(2) in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc(2) was further extended to an LM-like molecule. Complementation of C. glutamicum::pimB with Cg-pimB and Mt-pimB led to the restoration of LM biosynthesis. As a result, Cg-PimB, which we have assigned as MgtA, is now clearly defined as a GDP-mannose-dependent alpha-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc(2).

Adenosine Triphosphatases↗

PubMed2006

Resistance to Bacillus thuringiensis toxin in Caenorhabditis elegans from loss of fucose.

A mutation in the Caenorhabditis elegans bre-1 gene was isolated in a screen for Bacillus thuringiensis toxin-resistant (bre) mutants to the Cry5B crystal toxin made by B. thuringiensis. bre-1 mutant animals are different from the four other cloned bre mutants in that their level of resistance is noticeably lower. bre-1 animals also display a significantly reduced brood size at 25 degrees C. Here we cloned the bre-1 gene and characterized the bre-1 mutant phenotype. bre-1 encodes a protein with significant homology to a GDP-mannose 4,6-dehydratase, which catalyzes the first step in the biosynthesis of GDP-fucose from GDP-mannose. Injection of GDP-fucose but not fucose into C. elegans intestinal cells rescues bre-1 mutant phenotypes. Thus, C. elegans lacks a functional fucose salvage pathway. Furthermore, we demonstrate that bre-1 mutant animals are defective in production of fucosylated glycolipids and that bre-1 mutant animals make quantitatively reduced levels of glycolipid receptors for Cry5B. We finally show that bre-1 mutant animals, although viable, show a lack of fucosylated N- and O-glycans, based on mass spectrometric evidence. Thus, C. elegans can survive with little fucose and can develop resistance to crystal toxin by loss of a monosaccharide biosynthetic pathway.

Animals↗

PubMed2006

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation.

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.

Animals↗

PubMed2006

Mass spectrometric analysis of N- and O-glycosylation of tissues and cells.

Mass spectrometry has been at the forefront of glycobiological analysis for more than two decades. Recent advances in technology, coupled with increased global appreciation of the importance of the glycome in solving biological problems, have laid the foundations for an unprecedented increase in output. Under the auspices of major international consortia, significant progress is being made towards the development of integrated databases of specific cell and tissue N- and O-glycan profiles. Tools for the automation of both sample handling and data analysis are enabling a more concerted approach to glycan structural analysis. When combined with complementary transcriptomics and bioinformatics analyses, these tools will facilitate a new systems glycobiology approach to research.

Animals↗

PubMed2006

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans.

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.

Animals↗

PubMed2006

N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding.

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.

Amino Acid Sequence↗

PubMed2006

Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases.

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

Amino Acid Sequence↗

PubMed2006

Essential and mutually compensatory roles of {alpha}-mannosidase II and {alpha}-mannosidase IIx in N-glycan processing in vivo in mice.

Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N-glycosylation. alpha-Mannosidase II (MII) is a key enzyme converting precursor high-mannose-type N-glycans to matured complex-type structures. Previous studies showed that MII-null mice synthesize complex-type N-glycans, indicating the presence of an alternative pathway. Because alpha-mannosidase IIx (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N-glycan processing by generating MII/MX double-null mice. Some double-nulls died between embryonic days 15.5 and 18.5, but most survived until shortly after birth and died of respiratory failure, which represents a more severe phenotype than that seen in single-nulls for either gene. Structural analysis of N-glycans revealed that double-nulls completely lack complex-type N-glycans, demonstrating a critical role for at least one of these enzymes for effective N-glycan processing. Recombinant mouse MX and MII showed identical substrate specificities toward N-glycan substrates, suggesting that MX is an isozyme of MII. Thus, either MII or MX can biochemically compensate for the deficiency of the other in vivo, and either of two is required for late embryonic and early postnatal development.

Animals↗

PubMed2006

Protein glycosylation in Parelaphostrongylus tenuis--first description of the Galalpha1-3Gal sequence in a nematode.

The white-tailed deer is the definitive host of the parasitic nematode Parelaphostrongylus tenuis. This parasite also infects a wide variety of domesticated livestock, causing a debilitating neurologic disease. Glycoconjugates are becoming increasingly implicated in nematode strategies to maintain persistent infections in immunologically competent hosts. In this study, we have carried out detailed mass spectrometric analysis together with classical biochemical techniques, including western blotting and immunohistochemical staining with anticarbohydrate monoclonal antibodies and have shown that P. tenuis contains complex-type N-glycans with the antennae capped with Galalpha1-3Galbeta1-4GlcNAc sequence. By mimicking a vertebrate glycan, Galalpha1-3Gal may aid the parasite in evading immunological detection by the host. This is the first report of the Galalpha1-3Gal sequence in a nematode.

Animals↗

PubMed2006

N-Glycosylation of the MUC1 mucin in epithelial cells and secretions.

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.

Caco-2 Cells↗

PubMed2006

Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.

Amino Acid Sequence↗

PubMed2006

Molecular models for murine sperm-egg binding.

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of mouse eggs, known as the zona pellucida. Over the past decade, powerful genetic, biophysical, and biochemical techniques have been employed to gain new insights into this interaction. Evidence from these studies does not support either of two major models for binding first proposed over two decades ago. Two more recently established models suggest that protein-protein interactions predominate during this initial stage of fertilization. Another model proposes that about 75-80% of the murine sperm bound to zona pellucida under well defined in vitro conditions is carbohydrate dependent, with the remaining sperm bound via protein-protein interactions. Mounting evidence suggests that the carbohydrate sequences coating the murine egg could be employed as specific immune recognition markers. Continued investigation of this system may resolve many of these controversial findings and reveal novel functions for murine zona pellucida glycoproteins.

Animals↗

PubMed2006

Differential O-glycosylation of a conserved domain expressed in murine and human ZP3.

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.

Amino Acid Sequence↗

PubMed2006

Glycomics investigation into insulin action.

Defects in glycosylation are becoming increasingly associated with a range of human diseases. In some cases, the disease is caused by the glycosylation defect, whereas in others, the aberrant glycosylation may be a consequence of the disease. The implementation of highly sensitive and rapid mass spectrometric screening strategies for profiling the glycans present in model biological systems is revealing valuable insights into disease phenotypes. In addition, glycan screening is proving useful in the analysis of knock-out mice where it is possible to assess the role of glycosyltransferases and glycosidases and what function they have at the cellular and whole organism level. In this study, we analysed the effect of insulin on the glycosylation of 3T3-L1 cells and the effect of insulin resistance on glycosylation in a mouse model. Transcription profiling of 3T3-L1 cells treated with and without insulin revealed expression changes of several glycogenes. In contrast, mass spectrometric screening analysis of the glycans from these cells revealed very similar profiles suggesting that any changes in glycosylation were most likely on specific proteins rather than a global phenomenon. A fat-fed versus carbohydrate-fed mouse insulin resistant model was analysed to test the consequences of chronic insulin resistance. Muscle and liver N-glycosylation profiles from these mice are reported.

3T3-L1 Cells↗

PubMed2006

Glycomic studies of Drosophila melanogaster embryos.

With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galbeta1-4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galbeta1-3GalNAc sequence.

Animals↗

PubMed2006

Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies.

Over the past decade, rapid, high-sensitivity mass spectrometric strat-egies have been developed and optimized for screening for the types of N- and O-glycans present in a diverse range of biological material, including secretions, cell lines, tissues, and organs. These glycomic strategies are based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass fingerprinting of permethylated derivatives, combined with electrospray (ES) or MALDI tandem mass spectrometry (MS/MS) sequencing and gas chromatography (GC)-MS linkage analysis, complemented by chemical and enzymatic degradations. Protocols for these methods are described in the first part of this chapter. Glycomic experiments yield large volumes of MS data, and interpretation of the resulting spectra remains a time-consuming bottleneck in the process. In the second part of this chapter, we describe the use and operation of a mass spectral viewer program capable of displaying and automatically labeling spectra arising from MALDI fingerprinting of N-glycans.

Amino Acid Sequence↗

PubMed2006

Bacterial glycoproteomics.

Glycosylated proteins are ubiquitous components of eukaryote cellular surfaces, where the glycan moieties are implicated in a wide range of cell-cell recognition events. Once thought to be restricted to eukaryotes, glycosylation is now being increasingly reported in prokaryotes. Many of these discoveries have grown from advances in analytical technologies and genome sequencing. This review highlights the capabilities of high-sensitivity mass spectrometry for carbohydrate structure determination of bacterial glycoproteins and the emergence of glycoproteomic strategies that have evolved from proteomics and genomics for the functional analysis of bacterial glycosylation.

Bacteria↗