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1.
Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.  相似文献   

2.
O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.  相似文献   

3.
Wang H  Zhang W  Zhao J  Zhang L  Liu M  Yan G  Yao J  Yu H  Yang P 《Journal of Proteomics》2012,75(4):1375-1385
The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn157 for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.  相似文献   

4.
Protein glycosylation is widespread throughout all three domains of life. Bacterial protein N-glycosylation and its application to engineering recombinant glycoproteins continue to be actively studied. Here, we focus on advances made in the last 2 years, including the characterization of novel bacterial N-glycosylation pathways, examination of pathway enzymes and evolution, biological roles of protein modification in the native host, and exploitation of the N-glycosylation pathways to create novel vaccines and diagnostics.  相似文献   

5.
Modifications in cell surface glycosylation affecting cell adhesion are common characteristics of transformed cells. This study characterizes the N-glycosylation profile of E-cadherin in models of canine mammary gland adenoma and carcinoma evaluating the importance of these glycosylation modifications in the malignant phenotype.Our results show that the pattern of E-cadherin N-glycosylation in mammary carcinoma is characterized by highly branched N-glycans, increase in sialylation and an expression of few high mannose structures. Detailed mass spectrometry analysis demonstrated a new N-glycosylation site containing a potential complex type N-glycan in E-cadherin from a mammary carcinoma cell line.Our study demonstrates the importance of E-cadherin N-glycans in the process of tumor development and in the transformation to the malignant phenotype.  相似文献   

6.
Mucin-type O-glycosylation is an evolutionarily conserved protein modification present on membrane-bound and secreted proteins. Aberrations in O-glycosylation are responsible for certain human diseases and are associated with disease risk factors. Recent studies have demonstrated essential roles for mucin-type O-glycosylation in protein secretion, stability, processing, and function. Here, we summarize our current understanding of the diverse roles of mucin-type O-glycosylation during eukaryotic development. Appreciating how this conserved modification operates in developmental processes will provide insight into its roles in human disease and disease susceptibilities.  相似文献   

7.
Controlled protein remodeling with O-linked glycans has been limited by our incomplete understanding of the process of glycosylation. Here we describe a secretable fibroblast growth factor (FGF) with multiple mucin-type O-glycans produced by introducing a minimum pentapeptide glycosylation unit in a decarepeat format at its N- or C-terminus. Expressed in Chinese hamster ovary cells, chemical and biochemical analyses of the resultant proteins (Nm10-FGF and Cm10-FGF, respectively) demonstrated that all O-glycosylation units were glycosylated and the dominant structure was sialylated Gal[1–3]GalNAc. This indicates that minimum O-glycosylation unit in multirepeat format serves as a remarkably efficient acceptor in CHO cells. The Nm10-FGF and Cm10-FGF proteins maintained the mitogenic activity to vascular endothelial cells. In addition, intact Cm10-FGF and its desialylated form interacted with several lectins in the same way as mucin-type glycoproteins. The intact Cm10-FGF with multiple sialylated O-glycans exhibited a longer lifetime in circulating blood, whereas the Cm10-FGF with desialylated O-glycans exhibited a shorter lifetime than the deglycosylated form of Cm10-FGF. Our approach would thus appear to be highly effective for engineering neoglycoproteins, the characteristics of which are determined by their multiple mucin-type O-glycans.  相似文献   

8.
Esophagus squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors in the world. Our previous data demonstrates that oncoprotein MUC1 is related with metastasis and poor outcome of ESCC. However, alteration of MUC1 in ESCC remains unclear. Using ONCOMINE and COSMIC databases, we analyzed MUC1 gene copy numbers and gene mutations and found that MUC1 had high expression level but few gene mutations in ESCC. Further study of ESCC samples indicated that MUC1 O-glycosylation levels were higher in tumor tissues than that in para-carcinoma tissues in 10 of 14 pairs of ESCC samples. Moreover, we verified a potential link between MUC1 O-glycosylation and C1GALT1, which was further supported by IHC analysis on 38 ESCC and 19 para-carcinoma samples. More importantly, co-expression of MUC1 Oglycosylation and C1GALT1 presented positive correlations with both lymph node metastasis and survival time of ESCC patients. Our work collectively indicates that C1GALT1 is associated with O-glycosylated MUC1 in ESCC, not only suggesting a diagnostic significance of C1GALT1 and MUC1 O-glycosylation in ESCC, but also opening novel insights into targeting C1GALT1 and MUC1 O-glycosylation to suppress ESCC cells metastasis in patients.  相似文献   

9.
Human airway mucins represent a very broad family of polydisperse high molecular mass glycoproteins, which are part of the airway innate immunity. Apomucins, which correspond to their peptide part, are encoded by at least 6 different mucin genes (MUC1, MUC2, MUC4, MUC5B, MUC5AC and MUC7). The expression of some of these genes (at least MUC2 and MUC5AC) is induced by bacterial products, tobacco smoke and different cytokines.Human airway mucins are highly glycosylated (70–80% per weight). They contain from one single to several hundred carbohydrate chains. The carbohydrate chains that cover the apomucins are extremely diverse, adding to the complexity of these molecules. Structural information is available for more than 150 different O-glycan chains corresponding to the shortest chains (less than 12 sugars).The biosynthesis of these carbohydrate chains is a stepwise process involving many glycosyl- or sulfo-transferases. The only structural element shared by all mucin O-glycan chains is a GalNAc residue linked to a serine or threonine residue of the apomucin. There is growing evidence that the apomucin sequences influence the first glycosylation reactions. The elongation of the chains leads to various linear or branched extensions. Their non-reducing end, which corresponds to the termination of the chains, may bear different carbohydrate structures, such as histo-blood groups A or B determinants, H and sulfated H determinants, Lewis a, Lewis b, Lewis x or Lewis y epitopes, as well as sialyl- or sulfo- (sometimes sialyl- and sulfo-) Lewis a or Lewis x determinants. The synthesis of these different terminal determinants involves three different pathways with a whole set of glycosyl- and sulfo-transferases.Due to their wide structural diversity forming a combinatory of carbohydrate determinants as well as their location at the surface of the airways, mucins are involved in multiple interactions with microorganisms and are very important in the protection of the underlying airway mucosa.Airway mucins are oversulfated in cystic fibrosis and this feature has been considered as being linked to a primary defect of the disease. However, a similar pattern is observed in mucins from patients suffering from chronic bronchitis when they are severely infected. Airway mucins from severely infected patients suffering either from cystic fibrosis or from chronic bronchitis are also highly sialylated, and highly express sialylated and sulfated Lewis x determinants, a feature which may reflect severe mucosal inflammation or infection.These determinants are potential sites of attachment for Pseudomonas aeruginosa, the pathogen responsible for most of the morbidity and mortality in cystic fibrosis, and the expression of the sulfo- and glycosyl-transferases involved in their biosynthesis is increased by TNF.In summary, airway inflammation may simultaneously induce the expression of mucin genes (MUC2 and MUC5AC) and the expression of several glycosyl- and sulfo-transferases, therefore modifying the combinatory glycosylation of these molecules.  相似文献   

10.
Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.  相似文献   

11.
Notch signaling is essential for cell-fate specification in metazoans, and dysregulation of the pathway leads to a variety of human diseases including heart and vascular defects as well as cancer. Glycosylation of the Notch extracellular domain has emerged as an elegant means for regulating Notch activity, especially since the discovery that Fringe is a glycosyltransferase that modifies O-fucose in 2000. Since then, several other O-glycans on the extracellular domain have been demonstrated to modulate Notch activity. Here we will describe recent results on the molecular mechanisms by which Fringe modulates Notch activity, summarize recent work on how O-glucose, O-GlcNAc, and O-GalNAc glycans affect Notch, and discuss several human genetic disorders resulting from defects in Notch glycosylation.  相似文献   

12.
A role for N-linked oligosaccharides on the biochemical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn83–Thr–Thr and Asn202–Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn83, Asn202, and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn202 may contribute to thermostability and catalysis.  相似文献   

13.
14.
Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglBCj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglBCj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.  相似文献   

15.
Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.  相似文献   

16.
Increasing cellular O-GlcNAc levels through pharmacological inhibition of O-GlcNAcase, the enzyme responsible for removal of the O-GlcNAc post-translational modification, is being increasingly used to aid in discerning the roles played by this form of intracellular glycosylation. Interestingly, two forms of O-GlcNAcase have been studied; a full-length isoform that is better characterized, and a shorter nuclear-localized variant, arising from failure to splice out one intron, which has not been as well characterized. Given the increasing use of O-GlcNAcase inhibitors as research tools, we felt that a clear understanding of how these inhibitors affect both isoforms of O-GlcNAcase is important for proper interpretation of studies making use of these inhibitors in cell culture and in vivo. Here we describe an enzymatic characterization of the nuclear variant of human O-GlcNAcase. We find that this short nuclear variant of O-GlcNAcase, which has the identical catalytic domain as the full-length enzyme, has similar trends in a pH-rate profile and Taft linear free energy analysis as the full-length enzyme. These findings strongly suggest that both enzymes use broadly similar transition states. Consistent with this interpretation, the short isoform is potently inhibited by several previously described inhibitors of full-length O-GlcNAcase including PUGNAc, NAG-thiazoline, and the selective O-GlcNAcase inhibitor NButGT. These findings contrast with earlier studies and suggest that studies using O-GlcNAcase inhibitors in cultured cells or in vivo can be interpreted with the knowledge that both these forms of O-GlcNAcase are inhibited when present.  相似文献   

17.
Mucus hypersecretion occurs as a consequence of the Th2 immune response in epithelia, yet it was not previously known whether the degree of O-glycosylation was modulated under such conditions. A colonic carcinoma cell line LS174T was used to assess the effect of interleukin (IL)-4 on the mRNA levels of eight pp-GalNAc-Ts. A three- to four-fold increase in pp-GalNAc-T1, T4, and T7 levels was observed. Lysates of untreated or IL-4-treated cells were examined for their ability to transfer GalNAc residues onto a peptide corresponding to the tandem repeat portion of human MUC2. The number of incorporated GalNAc residues was greater after incubation with lysates of IL-4-treated cells than with lysates of untreated cells. Mucin-like large glycoproteins secreted by IL-4-treated cells had higher binding capacity to PNA and VVA-B4 than those secreted by untreated cells. The results indicated that IL-4-treated LS174T cells are able to produce mucins with a higher degree of O-glycosylation than untreated counterparts.  相似文献   

18.
Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.  相似文献   

19.
The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI–mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.  相似文献   

20.
Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.  相似文献   

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