Glycobiology - Advance Access

Effects of N-glycosylation on the activity and localization of GlcNAc-6-sulfotransferase 1

Desko, M. M, Gross, D. A, Kohler, J. J — 2009-07-01

N-Acetylglucosamine-6-sulfotransferase-1 (GlcNAc6ST-1) is a Golgi-resident glycoprotein that is responsible for sulfation of the l-selectin ligand on endothelial cells. Here, we report the sites at which GlcNAc6ST-1 is modified with N-linked glycans and the effects that each glycan has on enzyme activity, specificity, and localization. We determined that glycans are added at three of four potential N-linked glycosylation sites: N196, N410, and N428. The N428 glycan is required for the production of sulfated cell surface glycans: cells expressing a mutant enzyme lacking this glycan were unable to sulfate the sialyl Lewis X tetrasaccharide or a putative extended core 1 O-linked glycan. The N196 and N410 glycans differentially affect sulfation of two different substrates: cells that express an enzyme lacking the N410 glycan are able to sulfate the sialyl Lewis X substrate, but produce reduced levels of a sulfated peripheral lymph node addressin epitope and cells that express an enzyme lacking the N196 glycan are able to produce a sulfated peripheral lymph node addressin epitope, but are impaired in their ability to sulfate sialyl Lewis X. The glycans’ effects on enzyme activity may be mediated, in part, by changes in enzyme localization, while most mutants that lacked glycans localized normally within the Golgi, the N428A mutant, and a mutant lacking all glycans were also found to localize ectopically. Altered trafficking of mutants may be associated with the mechanisms by which misglycosylated enzyme is degraded.

Role of N-glycosylation of the SEA module of rodent Muc3 in posttranslational processing of its carboxy-terminal domain

2009-06-26

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two or three N-glycans, but was partially inhibited when lacking four, five and six N-glycans. In all, 1.99%, 48.34%, 85.03% and 72.98% of the products from N3457A, N34567A, N134567A and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

Galectin-1 stimulates monocyte chemotaxis via the p44/42 MAP kinase pathway and a Pertussis toxin sensitive pathway

Malik, R. K. J., Ghurye, R. R., Lawrence-Watt, D. J., Stewart, H. J. S. — 2009-06-26

Galectin-1, the prototype of a family of β galactoside-binding proteins, has been implicated in a wide variety of biological processes. Data presented herein show that galectin-1stimulates monocyte migration in a dose dependent manner but is not chemotactic for macrophages. Galectin-1 induced monocyte chemotaxis is blocked by lactose and inhibited by anti galectin-1 antibody but not by non specific antibodies. Furthermore galectin-1 mediated monocyte migration was significantly inhibited by MEK inhibitors in a rapid, time dependent manner suggesting that MAP kinase pathways are involved in galectin-1. Migration was also almost completely blocked by pertussis toxin implying G protein involvement in the galectin-1 induced chemotaxis.

These results demonstrate a role for galectin-1 in monocyte chemotaxis which differs from galectin-3 in that macrophages are non responsive. Furthermore our observations suggest that galectin-1 may be involved in chemoattraction at sites of inflammation in vivo and may contribute to disease processes such as atherosclerosis

Heterodisaccharide 4-O-(N-acetyl-{beta}-D-glucosaminyl)-D-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes

2009-06-24

Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)₂ from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)₂. In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.

A simple micro-method for determining precise oligosaccharidic specificity of mannose-binding lectins

Debray, H., Coddeville, B., Bomfim, L. R., Ramos, M. V. — 2009-06-19

A simple and inexpensive method was developed to rapidly define the specificity of mannose-specific lectins toward oligomannoside-type structures. The method involved the interaction of a mixture of N-[¹⁴C]-acetylated glycoasparagines, prepared by exhaustive pronase digestion of bovine pancreatic ribonuclease B and N-[¹⁴C]-acetylation with [¹⁴C]-acetic anhydride and containing all the possible oligomannoside-type N-glycans, with the lectin immobilized on Sepharose-4B. After exhaustive desalting, the obtained fractions were separated by high performance thin layer chromatography on silica gel plates and visualized by autoradiography with intensifying-screen. As an example of the usefulness of this method, the fine specificity of artocarpin, the mannose-specificity lectin isolated from seeds of jackfruit (Artocarpus integrifolia) toward oligomannoside-type structures is presented. On the basis of such a determination, the best oligomannosidic ligand recognized by a mannose-specific lectin can be selected for studies of crystal structures of the lectin in complex with the defined ligand. Furthermore, some of these immobilized lectins, after definition of their precise specificities with the method, could represent valuable tools for the fractionation and characterization of oligomannose-type structures, present in complex mixtures.

The specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry

Goto-Inoue, N., Hayasaka, T., Zaima, N., Setou, M. — 2009-06-19

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid called seminolipid. The galactosylation of the molecule is catalyzed by UDP-galactose: ceramide galactosyltransferase (CGT). Disruption of the CGT gene in mice results in male infertility due to the arrest of spermatogenesis, indicating that seminolipid plays an important role in reproductive function. Seminolipid molecules can be assigned to different molecular species based on the fatty acid composition. In this report, we investigated the localizations of the molecular species of seminolipid by imaging mass spectrometry and demonstrated that major molecule (C16:0-alkyl-C16:0-acyl) was expressed throughout the tubules, some (C16:0-alkyl-C14:0-acyl and C14:0-alkyl-C16:0-acyl) were predominantly expressed in spermatocytes and the other (C17:0-alkyl-C16:0-acyl) was specifically expressed in spermatids and spermatozoa. This is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation.

A novel role for Gtb1p in glucose trimming of N-linked glycans

Quinn, R. P., Mahoney, S. J., Wilkinson, B. M., Thornton, D. J., Stirling, C. J. — 2009-06-19

Glucosidase II (GluII) is a glycan trimming enzyme active on nascent glycoproteins in the endoplasmic reticulum (ER). It trims the middle and innermost glucose residues (Glc2 and Glc1) from N-linked glycans. The monoglucosylated glycan produced by the first GluII trimming reaction is recognised by calnexin/calreticulin and serves as the signal for entry into this folding pathway. GluII is a heterodimer of and β subunits corresponding to yeast Gls2p and Gtb1p respectively. While Gls2p contains the glucosyl hydrolase active site, the Gtb1p subunit has previously been shown to be essential for the Glc1 trimming event. Here we demonstrate that Gtb1p also determines the rate of Glc2 trimming. In order to further dissect these activities we mutagenised a number of conserved residues across the protein. Our data demonstrate that both the MRH and G2B domains of Gtb1p contribute to the Glc2 trimming event but that the MRH domain is essential for Glc1 trimming.

The {alpha}-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

Miller, M. C., Klyosov, A., Mayo, K. H. — 2009-06-18

Galectins are a sub-family of lectins, defined by their highly conserved β-sandwich structures and ability to bind to β-galactosides, like Gal-β(1->4)-Glc (lactose). Here, we used ¹⁵N-¹H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large (1->6)-D-galacto-β(1->4)-D-mannan Davanat (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (K_d) of 10 x 10^–6 M, compared to 260 x 10^–6 M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the β-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan, and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1-carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences.

Unusual accumulation of sulfated glycosphingolipids in colon cancer cells

2009-06-18

The structures of glycosphingolipids from highly purified colorectal cancer cells and normal colorectal epithelial cells of 16 patients have been analyzed in fine detail (Misonou, Y., et al. (2009) J. Proteome Res. 8(6):2990–3005). Further structural analyses demonstrated that colon cancer cells from 2 patients accumulated unusual glycosphingolipids which were not observed in either colorectal cancer cells or normal colorectal epithelial cells from the other patients. Mass spectrometry analyses revealed that the unusual structures include sulfated oligosaccharides. The structures of the glycosphingolipids of the cancer cells from these 2 cases were analyzed by methods which include enzymatic release of carbohydrate moieties, fluorescent labeling with aminopyridine and identification using two dimensional mapping, enzymatic digestion and mass spectrometry together with methanolysis and the use of newly synthesized sulfo-fucosylated oligosaccharides as standards. The colon cancer cells from one of the patients demonstrate a variety of oligosaccharides as major components which are sulfated at the C6 position of subterminal GlcNAc and at C3 positions of terminal galactose with or without sialylation or fucosylation. These include 6-sulfo Le^x, 6’-sialyl 6-sulfo lactosamine and 3’-sialyl 6-sulfo Le^x, in addition to sialylated or fucosylated derivatives of type-1 – type-2 hybrid oligosaccharides. The colon cancer cells from the other patient have 2 kinds of sulfated oligosaccharides, a 6-sulfo Le^x structure and a 3’-sulfo Le^x structure, as minor components. Taking into consideration the clinical features of the two patients, the biological significance of sulfated glycosphingolipids on cancer cells is discussed.

A newly generated functional antibody identifies Tn antigen as a novel determinant in cancer cell-lymphatic endothelium interaction.

2009-06-15

Malignant transformation of epithelial cells is frequently associated with the alteration of glycosilation pathways. Tn is a common Tumor-Associated Carbohydrate Antigen (TACA) present in 90% of human carcinomas and its expression correlates with metastatic potential and poor prognosis. Despite its relevance, the functional role of Tn in tumor biology has not been firmly established probably for the lack of appropriate experimental tools. Our aims were to produce highly reactive monoclonal antibodies (mAbs) against Tn making use of synthetically produced Tn and to test their usefulness for in vivo imaging as well as to define their potential functional activity in tumor cell spread. We immunized mice with Tn clustered on cationized BSA and screened the positive hybridomas with Tn-biotinylated alginate. ELISA and immunofluorescence assays revealed that the most reactive anti-Tn IgM mAb (2154F12A4) selectively recognized Tn on MCF7 breast cancer cell line, since its binding to the cell membrane was completely abolished by preincubation with purified Tn. Importantly, QDot 800-conjugated mAb injected in MCF7-tumor bearing mice specifically bound to primary tumor lesions as well as to metastases in lymph nodes. In addition, this mAb was able to inhibit cancer cell adhesion to lymphatic endothelium suggesting a novel involvement of Tn in the lymphatic dissemination of cancer cells and hypothesizing future applications in inhibiting lymphatic metastases.

Structural insights into what glycan arrays tell us about how glycan-binding proteins interact with their ligands

Taylor, M. E., Drickamer, K. — 2009-06-15

Screening of glycan arrays represents a powerful, high-throughput approach to defining oligosaccharide ligands for glycan-binding receptors, commonly referred to as lectins. Correlating results from such arrays with structural analysis of receptor-ligand complexes provides one way to validate the arrays. Using examples drawn from the family of proteins that contain C-type carbohydrate-recognition domains, this review illustrates how information from the arrays reflects the way that selectivity and affinity for glycan ligands is achieved. A range of binding profiles is observed, from very restricted binding to a small set of structurally similar ligands to binding of broad classes of ligands with related terminal sugars and even to failure to bind any of the glycans on an array. These outcomes provide insights into the importance of multiple factors in defining the selectivity of these receptors, including the presence of conformationally defined units in some oligosaccharide ligands, local and extended interactions between glycans and the surfaces of receptors, and steric factors that exclude binding of some ligands.

Two distinct {alpha}-L-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates

2009-06-11

Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2--l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama et al. 2004. J. Bacteriol. 186:4885–4893). Here, we identified a gene encoding a novel 1,3–1,4--l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 -l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released 1,3- and 1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing 1,2-fucosyl residue or on synthetic -fucoside (p-nitrophenyl--l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic -L-fucosidase. The transformant carrying afcA could utilize 2’-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading 1,2- and 1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

A mathematical model to derive N-glycan structures and cellular enzyme activities from mass spectrometric data

2009-06-08

Effective representation and characterization of biosynthetic pathways of glycosylation can be facilitated by mathematical modeling. This paper describes the expansion of a previously developed detailed model for N-linked glycosylation with the further application of the model to analyze MALDI-TOF mass spectra of human N-glycans in terms of underlying cellular enzyme activities. The glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes. The use of a molecular mass cutoff and a network pruning method typically limits the model size to about 10,000 glycan structures. This allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. A synthetic mass spectrum from model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with experiment. The result of this process is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of 19 enzymes. In addition a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. The method was applied to mass spectrometric data of normal human monocytes and monocytic leukemia (THP1) cells to derive glycosyltransferase activity changes underlying the differences in glycan structure between the normal and diseased cells. Model predictions could lead to a better understanding of the changes associated with disease states, identification of disease-associated biomarkers, and bioengineered glycan modifications.

Production of human {beta}-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of Ogataea minuta MNN4 gene

2009-06-08

Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human β-hexosaminidase A is a potentially therapeutic enzyme for GM2-gangliosidosis. Recombinant HexA has been produced by using the methylotrophic yeast Ogataea minuta as a host, and the purified enzyme was tested for its replacement effect on cultured fibroblasts derived from GM2-gangliosidosis patients. Although therapeutic effect was observed, in order to obtain higher therapeutic effect with little dose as possible, increased phosphorylation of recombinant β-hexosaminidase A N-glycans is suggested to be prerequisite. In the budding yeast Saccharomyces cerevisiae, overexpression of MNN4, which encodes a positive regulator of mannosylphosphate transferase, led to increased mannosylphosphate contents. In the present study, we cloned OmMNN4, a homologous gene to ScMNN4, based on the genomic sequence of O. minuta. We overexpressed the cloned gene under the control of the alcohol oxidase promoter in a β-hexosaminidase A-producing yeast strain. Structural analysis of pyridylamine-labeled N-glycans by high-performance liquid chromatography revealed that overexpression of MNN4 caused a three-fold increase in phosphorylated N-glycans of recombinant β-hexosaminidase A. Recombinant enzyme prepared from strains overexpressing OmMNN4 was more effectively incorporated into cultured fibroblasts and neural cells, and it more rapidly degraded the accumulated GM2-ganglioside as compared to the control enzyme. These results suggest that β-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.

Optimal and Consistent Protein Glycosylation in Mammalian Cell Culture

Hossler, P., Khattak, S. F., Li, Z. — 2009-06-03

In the biopharmaceutical industry, mammalian cell culture systems, especially Chinese hamster ovary (CHO) cells, are predominantly used for the production of therapeutic glycoproteins. Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein. Obtaining a consistent glycoform profile in production is desired due to regulatory concerns because a molecule can be defined by its carbohydrate structures. An optimal profile may involve a spectrum of product glycans that confers a desired therapeutic efficacy, or a homogeneous glycoform profile that can be systemically screened for. Studies have shown some degree of protein glycosylation control in mammalian cell culture, through cellular, media, and process effects. Studies upon our own bioprocesses to produce fusion proteins and monoclonal antibodies have shown an intricate relationship between these variables and the resulting protein quality. Glycosylation optimization will improve therapeutic efficacy and is an ongoing goal for researchers in academia and industry alike. This review will focus on the advancements made in glycosylation control in a manufacturing process, as well as the next steps in understanding and controlling protein glycosylation.

Catabolism of flocculosin, an antimicrobial metabolite produced by Pseudozyma flocculosa

Mimee, B., Labbe, C., Belanger, R. R. — 2009-06-03

Flocculosin is an unusual cellobiose lipid secreted by the yeast-like fungus Pseudozyma flocculosa as part of its biocontrol arsenal against other fungi. Recent observations have suggested that the fungus degrades flocculosin to use it as a nutrient source during periods of food limitation. In this work, we sought to identify the catabolic steps leading to the degradation of flocculosin and its subsequent use by P. flocculosa. To this end, we characterized the catabolism of flocculosin through identification of degradation intermediates in a deprived medium using mass spectrometry. As the pH of the medium increased, the molecule was quickly deacylated and lost its antimicrobial activity thereby explaining conflicting results concerning the antimicrobial activity of this class of glycolipid. Following removal of both acetyl groups and the short fatty acid chain under alkaline conditions, the molecule was quickly and completely metabolized by P. flocculosa. Protein purification of culture filtrates confirmed the presence of degradative enzymes produced by P. flocculosa. These enzymes were found to degrade 3,15-dihydroxy-hexadecyl cellobioside (DHC) but not the acylated molecule thus confirming the protective role of these groups against catabolism. These results are the first evidence of glycolipid degradation by the producing organism and suggest that flocculosin can be recycled by P. flocculosa as a nutrient in addition to protecting its ecological niche.

Establishment of a real-time analytical method for free oligosaccharide transport from the ER to the cytosol

Haga, Y., Totani, K., Ito, Y., Suzuki, T. — 2009-06-03

During N-glycosylation of proteins, significant amounts of free unconjugated glycans are also generated in the lumen of the endoplasmic reticulum (ER). These ER-derived free glycans are translocated into the cytosol by a putative transporter on the ER membrane for further processing. However, the molecular nature of the transporter remains to be determined. Here, we report the establishment of a novel assay method for free oligosaccharide transport from the ER lumen using chemically synthesized fluorescence-labeled N-glycan derivatives. In this method, fluorescence-labeled glycan substrates were encapsulated inside mouse liver microsomes, followed by incubation with the cytosol and a fluorescence-quenching agent (anti-fluorophore antibody). The rate of substrate efflux was then monitored in real time by the decrease in the fluorescence intensity. The present data clearly demonstrated that the oligosaccharide transport activity under the current assay conditions was both ATP- and cytosol-dependent. The transporter activity was also found to be glycan structure-specific, because free glucosylated glycans were unable to be transported out of the microsomes. This new assay method will be a useful tool for identifying the transporter protein on the ER membrane.

Mutational and Functional Analysis of Large in a Novel CHO Glycosylation Mutant

Aguilan, J. T., Sundaram, S., Nieves, E., Stanley, P. — 2009-05-21

Inactivating mutations of Large reduce the functional glycosylation of -dystroglycan (-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT) and the C-terminal domain is related to the human i blood group transferase β1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, -DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the β1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional -DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional -DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally-glycosylated -DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of -DG.

Glycosylation profiles of epitope - specific anti-ss-amyloid antibodies revealed by liquid chromatography - mass spectrometry

Perdivara, I., Deterding, L. J., Cozma, C., Tomer, K. B., Przybylski, M. — 2009-03-24

Alzheimer's disease (AD) is the most prevalent form of age-related neurodementia. The accumulation of ß-amyloid polypeptide (Aß) in brain is generally believed to be a key event in AD. The recent discovery of physiological ß-amyloid-autoantibodies represents a promising perspective for treatment and early diagnosis of AD. The mechanisms by which natural ß-amyloid autoantibodies prevent neurodegeneration are currently unknown. The aim of the present study was to analyze the N-linked glycosylation of a plaque-specific, monoclonal antibody (clone 6E10) relevant for immunotherapy of AD, in comparison to the glycosylation pattern of an Aβ-autoantibody isolated from an IgG source. Liquid chromatography in combination with tandem mass spectrometry was used to analyze the glycopeptides generated by enzymatic degradation of the antibodies reduced and alkylated heavy chains. The oligosaccharide pattern of the 6E10 antibody shows primarily core-fucosylated biantennary complex structures and, to a low extent, tri- and tetragalactosyl glycoforms, with or without terminal sialic acids. The glycans associated with the serum anti-Aß-autoantibodies are of the complex, biantennary type, fucosylated at the first N-acetyl glucosamine residue of the trimannosyl chitobiose core, and contain zero to two galactose residues, and zero to one terminal sialic acid, with or without bisecting N-acetyl glucosamine. Glycosylation analysis of the Aß-autoantibody performed at the peptide level revealed all four human IgG subclasses, with IgG₁ and IgG₂ as the dominant subclasses.