Alpha-Dystroglycan (αDG) possesses a mucin-like domain with multiple serine (S) and threonine (T) residues, that express O-linked mannosylated (O-Man) moieties that are then elongated by O-mannosyl-β1,2-N-acetylglucosaminyltransferase (POMGnT1) and other glycosyltransferases. Mutations in POMGnT1 result in truncated structures that cause muscle-eye-brain disease. In order to understand the acceptor specificity of POMGnT1, we have synthesized eight peptide sequences derived from the mucin-like domain of αDG (which spans from residues 316 to 489), each with one or multiple O-Man sites. These peptides were designated as M1 (residues 416-420 with O-Man at T418), M2 (residues 429-433 with O-Man sites at S430 and T431), M3 (residues 326-331 with O-man sites at T328 and T329), M4 (residues 411-416 with O-Man at T414), M5 (residues 461-466 with O-Man sites at T463 and T464), M6 (residues 480-487 with O-Man sites at T482, T483, T484, and S485), M7 (residues 419-427 with O-Man sites at T421 and T424), and M8 (residues 419-427 with O-Man sites at T421, T422, T423, and T424). These peptides were used as in vitro acceptors for recombinant POMGnT1 secreted by HEK-293 cells, and kinetic parameters (Km, Kcat, and Km/Kcat) were determined for each substrate. Results show a higher specificity of POMGnT1 for substrates M2, M3 and M4. Mass spectrometric analysis of the reaction products of the M2 and M3 substrates (each with two O-Man sites) show that only one mannosylation site is utilized by the enzyme. These results suggest that POMGnT1 has increased affinity for specific amino acid sequences in the mucin-like domain of αDG.

ST6Gal1 is one of the most studied enzymes of the CAZy Family 29 (GT29) glycosyltransferases. The enzyme has been shown to modulate CD22-mediated immune responses through the synthesis of its trisaccharide ligand product, Siaα2-6Galβ1-4GlcNAc (Sia6LacNAc). Despite such a critical role in immune function, no structural data is yet available for ST6Gal1 or any other CAZy GT29 family enzyme. A major limitation in the structural analysis of glycosylation enzymes, including ST6Gal1, is the large-scale expression and purification of the glycosylated enzymes in forms compatible with structural analysis.

We have successfully generated recombinant Pichia expression constructs using a modular approach to encode secreted forms of sialyltransferases. A construct containing the ST6Gal1 coding region was prepared in the Pichia vector pPIC-Z-αC and transformed into Pichia host strains. Controlled fermentation was used for expression of recombinant ST6Gal1 and the media composition and fermentation conditions were optimized in order to maximize recovery and the ¹⁵N labeling of recombinant enzyme. HSQC spectra of uniformly ¹⁵N labeled ST6Gal1 indicated that heterogeneous glycosylation may be a limiting factor for generation of a homogeneous enzyme preparation for NMR and X-ray crystallography. Glycan heterogeneity was subsequently eliminated by treating recombinant ST6Gal1 with Endo H followed by chromatography over ConA-Sepharose. Further characterization of deglycosylated ST6Gal1 by NMR indicates a flexible enzyme partially stabilized by substrate binding. A multiple isotope labeling strategy (²H, ¹³C and ¹⁵N) is underway focused on NMR resonance assignment and eventual structural determination of the recombinant enzyme (Supported by NIH grant RR005351).

The glycoprotein alpha-dystroglycan (alpha-DG) has generated much interest because of its presence as a key component of the dystrophin glycoprotein complex, particularly in organizing the extracellular matrix in muscle tissue, its rare feature of alpha-O-linked mannosyl protein glycosylation, and the relationship of defects in its glycosylation with muscular dystrophy pathologies. The sequential glycosylation steps, with initial O-mannosylation on some of the S and T residues followed by addition of GalNAcs and extension of glycans, provide an interesting system to probe relative conformational effects of the two modes of glycosylation and factors effecting selectivity of polypeptide GalNAc transferase enzymes. To investigate these, a series of mannosylated glycopeptides based on amino acid sequences from the central mucin-like portion of alpha-DG have been prepared. These provide well defined models for characterizing their properties as enzyme substrates and for their conformational study with NMR methods. Glycosylation mapping in the native glycoprotein indicates that O-Man and O-GalNAc residues occur in close proximity, and we are evaluating the ability of several ppGalNAc transferases to install GalNAcs in analogous glycopeptide contexts. In conformational analysis, we are refining the structure of mannosylated glycopeptides and comparing the results with an analogous construct with GalNAc instead of mannose. This combined effort is revealing the relative roles of the two substitutions in fostering the extended structure of the glycoprotein backbone in ?-DG, and contributing to the understanding of structure-function relationships in the processing of the glycoprotein and in its ultimate properties.

The enzymes produced by fungi that degrade pectin play crucial roles in both agriculture and industry. While pectin degrading enzymes are industrially beneficial and their production represents a multi-billion dollar industry, agricultural crop loss due to phytopathogenic fungi is a worldwide problem. Many fungi use endopolygalacturonases (EPGs) to hydrolyze the cell wall polysaccharide homogalacturonan as one of the first steps in invasion. Plant defense mechanisms directed toward EPGs have evolved that utilize polygalacturonase-inhibiting proteins (PGIPs) to alter the activity of the EPG. The mode of action of a particular fungal EPG and its inhibition by PGIPs may be critical factors in determining whether the fungus is a viable pathogen. At the same time, the interaction between EPGs and PGIPs can be affected by different factors such as pH and substrate. We are using surface plasmon resonance to study the specificity and kinetics of the interactions between bean polygalacturonase-inhibiting protein (PGIP) and different fungal endopolygalacturonases (EPGs) in the presence or absence of homogalacturan. Results show that the presence of substrate has a moderate to strong effect on the EPG/PGIP interaction, and that the level of that effect is dependent on the exact EPG/PGIP pairing. Differential proteolysis/MS experiments have allowed us to identify the probable sites of EPG/PGIP interaction.

The Drosophila embryo expresses a family of related N-linked glycan structures, known as HRP-epitopes, that are enriched in neural tissue and carry Fuc linked α3 to the reducing terminal GlcNAc of the chitobiose core. In a screen for mutations that affect tissue specific glycosylation, we generated a new mutation, named sugar-free frosting (sff), that specifically abolishes almost all HRP-epitope expression. Our single mutant allele of sff (sff^B22) is semi-viable. Homozygous adults display a deficit in geotaxis, which is rescued by acute administration of drugs that affect biogenic amine metabolism. We have noticed that specific genetic backgrounds, particularly those that include mutations in the white gene (w), affect viability and the efficacy of drug treatment in sff^B22. Therefore, characterization of the N-linked glycan profile of sff^B22 embryos was performed in two different genetic backgrounds, either wild-type or mutant for white (w⁺, and w¹¹¹⁸, respectively). In either background, sff^B22 reduces both α3-linked Fuc and the prevalence of some minor complex glycans, while other changes reflect the genetic background. In parallel, we compared the glycan profiles of wild-type (OreR) and w¹¹¹⁸ embryos to provide a background for assessing gene-specific effects. The predominant glycans of both genotypes, primarily pauci- and high-mannose structures, exhibit similar prevalences. However, significant differences were apparent among less prevalent, complex glycans. Therefore, differences in the genetic backgrounds of nominally wild-type strains can influence the flux and nature of glycan processing and should be taken into account when interpreting mutant phenotypes. Supported by funding from NIH/NIGMS.

One of the major challenges in the –omics field is the development of technologies that allow for quantitative analysis between samples. In proteomics, stable isotope approaches, such as SILAC, have been developed to address this need. Here we report a methodology that takes advantage of stable isotope labeling of glycans in cell culture for performing relative quantitative glycomics. This methodology termed IDAWG, isotopic detection of aminosugars with glutamine, relies on the hexosamine biosynthetic pathway that uses the side-chain of glutamine as its sole donor source of nitrogen for aminosugars in the production of sugar nucleotides. Thus, introduction of heavy glutamine (¹⁵N) into Gln-free media allows for all aminosugars to become labeled and shifted in mass by +1 dalton. Here we demonstrate that this methodology allows for rapid and nearly complete incorporation of ¹⁵N into GlcNAc, GalNAc, and sialic acids of N-linked and O-linked glycans in various mammalian cell culture systems. Besides aiding in the assignment of structures via LC-MSⁿ approaches, this method allows us to determine whether the glycans isolated from a sample result from cellular processes or serum glycoproteins. Importantly, this method also allows us to compare in a quantitative manner the glycans between two cell populations. Furthermore, half-life studies can be performed on glycan structures by switching a cell population from heavy to light labeling conditions and harvesting and analyzing the glycans by LC-MSⁿ approaches at multiple time points afterwards. Thus, the IDAWG approach is an easily applied and powerful new tool in the glycomics toolbox.

The O-GlcNAc modification, a ubiquitous and dynamic intracellular glycosylation on the serine and threonine residues of polypeptides, serves as a negative regulator of the insulin-mediated PI3K/Akt signaling cascade with a concomitant defect at or upstream of Akt activation in mammalian cell culture models. This defect results in insulin resistance, which precedes and is the hallmark of type II diabetes, in adipocytes. While activation of the PI3K/Akt pathway is a universal master switch of various receptor tyrosine kinase (RTK)-mediated signaling pathways, the impact of elevated O-GlcNAc modification on RTK pathways other than the insulin pathway has yet to be elucidated. In this study, we examined the activation status of the PI3K/Akt pathway via two other predominant RTK pathways mediated by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in 3T3-L1 adipocytes in response to global elevation of O-GlcNAc levels. Our results showed that the inhibition of Akt activation via elevated O-GlcNAc levels is exclusively an insulin signaling-specific phenomenon. Given that increasing O-GlcNAc levels does not affect the autophosphorylation capability of the insulin receptor, our results strongly suggest that the IRS proteins, which are O-GlcNAc modified, are the target for functional O-GlcNAc modification and fine-tuning of the insulin signal transduction pathway. Current studies are aimed at confirming these results using genetic means to alter O-GlcNAc levels in relevant cell lines for the aforementioned and other RTK pathways. As well, mapping and mutagenesis of O-GlcNAc sites on IRS proteins for functional studies is underway.

ST6 beta-galactosamide alpha-2, 6-sialyltranferase ? (ST6 Gal ?) enzyme is responsible for the transfer of sialic acid to the 6-hydroxyl group of galactose. The carbohydrate parts in ST6 play an important role for the function of the enzyme. Therefore analysis of the carbohydrate structure is necessary for characterization of ST6 Gal ?. Sialylation by ST6 Gal ? occur mainly at the galactose residue of the terminal Gal-GlcNAc of either N-linked or O-linked carbohydrates. The N-glycans were released by treatment with PNGase F and subsequently permethylated prior to MS analysis. Complex mixture of N-glycans was detected and the main component was found to be the HexNAc5.Hex4.NeuAc2.Fuc1 structure. The above structure corresponded to an unusual fucosylated biantennary structure with two sialic acids and a HexNAc-HexNAc structure on one of the biantennary arms. The glycoprotein was sequentially digested with ? 2-3 Neuraminidase, ? N-acetylhexosaminidase, ? 1-2,3 mannosidase, and ? 1-6 mannosidase followed by permethylation. The structure of derivatized glycan residues were elucidated by MALDI-TOF-MS and ESI-MS/MS. The presence of GalNAc-GlcNAc structure on the biantennary arm instead of the usual Gal-GlcNAc was confirmed by specific exoglycosidase digestions. The linkage of the GalNAc-GlcNAc-residues to the ?-mannose (either 1-3 or 1-6 linkage) is being confirmed. Preliminary data indicates that the GalNAc-GlcNAc structure is attached to the 3-arm..

Modification of mammalian proteins by O-Mannose-initiated glycans has received increased attention recently due to the implicated role of these structures in congenital muscular dystrophy. Multiple forms of these diseases result from defects in glycosyltransferases involved in the addition and extension of O-Mannose, including Muscle-Eye-Brain disease (MEB) that is caused by mutations in the gene encoding protein O-mannose β-1,2-glucosaminyltransferase (POMGnT1). We have characterized via LC-MSⁿ the permethylated O-linked glycans released by reductive elimination from brain proteins of wildtype and POMGnT1 knockout mice. As expected, ~30% of the observed O-linked glycans released from wildtype brain proteins are O-Man initiated and elongated O-Man glycans were not detected in the POMGnT1 knockout brains. To date, the only well characterized O-mannose modified mammalian protein is alpha-dystroglycan. Since the O-Man-initiated structures are so important in disease processes, we have developed a strategy for identifying the full complement of O-Man modified proteins. As proof of principal, a synthetic alpha-dystroglycan derived O-Man modified peptide has been extended with azido-modified GlcNAc using recombinant, purified POMGnT1 and UDP-GlcNAz. Staudinger ligation was subsequently used to attach a FLAG tag to this peptide for affinity purification. An alternative approach was developed in which GlcNAc was added with POMGnT1 to the O-Man peptide followed by addition of a keto-modified Gal using a mutant GalT. This keto-sugar can then be reacted with aminoxybiotin for affinity purification. A comprehensive identification of the full diversity of O-Man modified proteins will result from applying these tagging strategies to glycopeptides generated from POMGnT1 knockout brains.

The aim of the present study is to determine the structure and positions of glycosylation sites on the Drosophila sialyltransferase protein, DSiaT. The DSiaT represents the first characterized sialyltransferase in the protostome lineage of animals. This sialyltransferase is closely related to the ST6Gal family of vertebrate sialyltransferases, which indicates that DSiaT may represent the most evolutionary ancient type of matazoan sialyltransferases. The DSiaT-Protein A fusion protein has been expressed in Drosophila S2 culture cells and purified as described earlier (Koles et al. 2004, JBC 279: 4346-4357). The DSiaT from a Coomassie Blue stained polyacrylamide gel were excised and then performed tryptic digestion in gel. The N-linked oligosaccharides were released from the glycopeptides extracted from gel pieces. The released N-glycans are permethylated and then analyzed by both MALDI-MS and NSI-MS/MS. To capture all glycans, automated MS/MS (total ion mapping) was also performed. MALDI-MS spectrum revealed that the main glycan of DSiaT has a fucosylated high-mannnose structure, Man3GlcNAc2Fuc1 (m/z = 1345). Another glycan with a high-mannose structure, Man3GlcNAc2 (m/z = 1171) was also detected as a minor component. In addition, the minor ions observed in the NSI-MS spectrum included more than ten glycans with high mannose structures. The all glycans found in NSI-MS spectrum were confirmed by NSI-MS/MS. The sites of N-linked glycosylation will also be determined using LC-MS analysis of the 18O labeling of asparagines residue prepared from the DSiaT glycoprotein.

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. Genetic, biochemical, and pathophysiologic studies in vertebrate and non-vertebrate species have repeatedly demonstrated the importance of O-linked glycans, including O-Man, O-Fuc, O-GlcNAc, O-Glc, as well as O-GalNAc initiated mucin-type structures. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have optimized methodology for analyzing the full complement of O-linked glycans in Drosophila embryos. Through multi-dimensional mass spectrometric analysis of permethylated glycans, following release from glycoprotein by reductive β-elimination, we detect both novel and previously reported O-glycans. The Core 1 mucin-type disaccharide (Galβ1,3GalNAc) is the predominant glycan in the total profile. HexNAcitol, Hexitol, xylosylated Hexitol, and branching extension of Core 1 with HexNAc (to generate Core 2 glycans) are also evident. After Galβ1,3GalNAc, the next most prevalent glycans are a mixture of novel, isobaric, linear and branched forms of a glucuronyl Core 1 disaccharide. Other less prevalent glycans are also extended with HexA, including Core 2 and O-Fuc structures. Although the expected disaccharide product of the Fringe glycosyltransferase (GlcNAcβ1,3Fucitol) is barely detectable in whole embryos, MSⁿ fragmentation and exoglycosidase sensitivity define a more prevalent, novel glucuronyl trisaccharide as GlcNAcβ1,3(GlcAβ1,4)Fucitol. Therefore, as described for Notch protein modification in vertebrates, Fringe-extended O-Fuc in Drosophila is a substrate for further elaboration. Full characterization of the Drosophila O-linked glycome, coupled with analysis of tissue- and stage-specific expression in wild-type and relevant mutant backgrounds, now provides an enriched context for assessing specific glycan functions.

Glycosylation is thought to play an important role in the development and function of the nervous system, given the immense number of highly specific synaptic connections in the brain. Even though a large number of glycoproteins have been studied in the CNS of many organisms, very little information is available about the functional role of their N- or O-glycans. In an attempt to fill this gap, we have initiated a large-scale identification of N-glycosylated glycoproteins and their N-glycan attachment sites from the fruit-fly brain. Using a combination of lectin-affinity chromatography steps and LC-MS/MS analysis we identified a total of 205 glycoproteins carrying at least one N-linked carbohydrate chain and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. N-glycans, which were released and analyzed separately, were found to be dominated by oligo- and paucimannosidic structures, although a monoantennary sialylated glycan was also detected. The wealth of information gathered in this study should significantly facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the CNS of Drosophila.

The functional effects of glycosylation are controlled by the coordinated expression of glycosyltransferases and the regulated expression of particular glycoprotein acceptors. O-mannosyl-linked glycosylation is abundant within the central nervous system, yet very few glycoproteins with this glycan modification have been identified, and the functional roles of this glycan modification have not been defined. Congenital diseases with significant neurological defects arise from inactivating mutations found within the glycosyltransferases that act early in this pathway. We are studying the glycosyltransferase known as GnT-Vb that is responsible for elongating O-mannosyl-linked glycans allowing for the addition of further glycan elaborations such as the HNK-1 epitope. We have identified a key substrate of GnT-Vb and provide evidence that GnT-Vb-mediated glycosylation of this receptor alters neuronal signal transduction that influences cell-cell interactions and migration in the developing nervous system.