N-linked glycosylation is required for nicotinic receptor assembly but not for subunit associations with calnexin

We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation.     

Role of nonenzymatic glycosylation in experimental cataract formation

The relevance of nonenzymatic glycosylation of lens proteins to cataract formation was studied in rats on a normal and high galactose diet, treated with and without sorbinil, an aldose reductase inhibitor. All galactosemic rats not receiving sorbinil had cataracts; none receiving sorbinil had cataracts. Lens homogenate was treated with a 200 fold molar excess of [³H]-borohydride and the extent of glycosylation was estimated from radioactivity incorporation and quantitation of hexitol-lysine adduct after extensive dialysis. We found no differences in the radioactivity uptake nor the amounts of hexitol-lysine in the lenses of galactosemic rats treated with and without sorbinil. Thus, nonenzymatic glycosylation was not responsible for the sugar-induced cataracts.     

Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry

We have previously determined the carbohydrate structure of human recombinant erythropoietin [Sasaki, H., Bothner, B., Dell, A., & Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076]. The carbohydrate chains are distributed in three N-glycosylation sites and one O-glycosylation site. In order to examine the extent to which protein structure influences glycosylation, we have analyzed the saccharide structures at each glycosylation site (Asn24, Asn38, Asn83, and Ser126) of human recombinant erythropoietin. By high-performance liquid chromatography, we have succeeded in separation of glycopeptides containing different O-linked saccharides to the same peptide backbone. Fast atom bombardment mass spectrometry of the isolated glycopeptides combined with Edman degradation allowed us to elucidate the composition of glycopeptides and the amino acid attachment site. The analysis of glycopeptides and saccharides by fast atom bombardment mass spectrometry and high-performance liquid chromatography provided the following conclusions on N-glycans: (1) saccharides at Asn24 are heterogeneous and consist of biantennary, triantennary, and tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (2) saccharides at Asn38 mainly consist of well-processed saccharides such as tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (3) saccharides at Asn83, on the other hand, are homogeneous in the backbone structure and are composed mainly of tetraantennary without N-acetyllactosaminyl repeats. It was also noted that saccharides at Asn24 are much less sialylated than those at Asn38, although these two glycosylation sites are close to each other. These results clearly indicate that the protein structure and, possibly, the carbohydrate chain at the neighboring site greatly influence glycosylation of a given glycosylation site.     

鸡α干扰素在毕赤酵母中的表达及糖基化对其表达的影响

目的:实现鸡α干扰素(ChIFNα)在毕赤酵母GS115中的表达,并考察糖基化对其表达的影响。方法:人工合成按照毕赤酵母偏好密码子优化的ChIFNα基因,克隆至分泌表达载体pPIC9,电转到毕赤酵母GS115中进行表达。利用定点突变对ChIFNα基因序列中4个糖基化位点进行缺失,SDS-PAGE分析N-糖基化对毕赤酵母表达ChIFNα的影响。结果:ChIFNα在GS115中获得了表达,在摇瓶发酵条件下,表达量为35.2 mg/L;构建了缺失1~4个糖基化位点的4个突变体,在GS115中实现了表达,分析表达结果显示,与未突变的ChIFNα对照相比,突变体的糖基化程度和表达量都有大幅度下降。结论:N-糖基化对于毕赤酵母表达ChIFNα具有重要影响,无糖基化的ChIFNα在毕赤酵母中表达量极低。     

Golgi glycosylation and human inherited diseases

The Golgi factory receives custom glycosylates and dispatches its cargo to the correct cellular locations. The process requires importing donor substrates, moving the cargo, and recycling machinery. Correctly glycosylated cargo reflects the Golgi's quality and efficiency. Genetic disorders in the specific equipment (enzymes), donors (nucleotide sugar transporters), or equipment recycling/reorganization components (COG, SEC, golgins) can all affect glycosylation. Dozens of human glycosylation disorders fit these categories. Many other genes, with or without familiar names, well-annotated pedigrees, or likely homologies will join the ranks of glycosylation disorders. Their broad and unpredictable case-by-case phenotypes cross the traditional medical specialty boundaries. The gene functions in patients may be elusive, but their common feature may include altered glycosylation that provide clues to Golgi function. This article focuses on a group of human disorders that affect protein or lipid glycosylation. Readers may find it useful to generalize some of these patient-based, translational observations to their own research.     

Glucosamine itself mediates reversible inhibition of protein glycosylation. A study of glucosamine metabolism at inhibitory concentrations in influenza-virus-infected cells

The metabolism of glucosamine in chick embryo fibroblasts was studied at different concentrations of the amino sugar added to the culture medium. In glucose-containing medium the well-known metabolites, UDP-N-acetylglucosamine, N-acetylglucosamine 6-phosphate and N-acetylglucosamine, are detectable after inhibition of glycosylation resulting from glucosamine treatment. Especially when the cells were infected with influenza virus, high intracellular concentrations of non-metabolized glucosamine are demonstrable in addition. Removal of the inhibitor from the medium results in release of the block of influenza virus glycoprotein glycosylation within 10 min. The onset of glycosylation is paralleled by a rapid reduction of intracellular levels of glucosamine without significant changes in the concentration of its metabolites. Furthermore, concentrations of GDP-mannose, UDP-glucose, and UDP-galactose remain constant for at least 30 min after reversal of the block. It is concluded that glucosamine as such exerts its effect on glycosylation, rather than one of its metabolites being responsible for this effect.     

Effect of altered glycosylation on the structure of the I-like domain of beta1 integrin: a molecular dynamics study

Glycosylation plays an important role in the regulation of integrin function. Molecular mechanisms underlying the effects of altered glycosylation on beta1 integrin structure and function are still largely unknown. In this study, we used a molecular modeling approach to study the effects of altered glycosylation, with alpha2-6 sialic acid and without alpha2-6 sialic acid, on the structure of the I-like domain of the beta1 integrin. Our results demonstrated that altered glycosylation affected the interactions between oligosaccharides and the I-like domain, which in turn changed the accessibility of the specificity-determining loop for ligand binding. Altered glycosylation caused significant conformational changes for most of the key functional regions of the I-like domain of beta1 integrin, including the metal ion-dependent adhesion site that contains a DLSYS motif, and other critical residues for ligand binding (Asn-224, Glu-229, Asp-233, Asp-267, and Asp-295). In addition, altered glycosylation caused significant movement of the alpha1 and alpha7 helices, which are important for the activation of beta1 integrin. The results from this study offered molecular mechanisms for the experimental observations that variant glycosylation regulates integrin function.     

Functional effects of coxsackievirus and adenovirus receptor glycosylation on homophilic adhesion and adenoviral infection

The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus.     

Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation

Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound beta-domain and a passenger domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several passenger domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. In the present study we describe the effect of glycosylation on the extracellular passenger domain of the Escherichia coli autotransporter Ag43alpha, which induces frizzy colony morphology and cell settling. We identify 16 glycosylation sites and suggest two possible glycosylation motifs for serine and threonine residues. Glycosylation stabilizes against thermal and chemical denaturation and increases refolding kinetics. Unexpectedly, glycosylation also reduces the stabilizing effect of Ca(2+) ions, removes the ability of Ca(2+) to promote cell adhesion, reduces the ability of Ag43alpha-containing cells to form bacterial amyloid and increases the susceptibility of the resulting amyloid to proteolysis. In addition, our results indicate that Ag43alpha folds without a stable intermediate, unlike pertactin, indicating that autotransporters may arrive at the native state by a variety of different mechanisms despite a common overall structure. A small but significant fraction of Ag43alpha can survive intact in the periplasm if expressed without the beta-domain, suggesting that it is able to adopt a protease-resistant structure prior to translocation across the membrane. The present study demonstrates that glycosylation may play significant roles in structural and functional properties of bacterial autotransporters at many different levels.     

A和C结构域糖基化位点对凝血八因子的分泌及活性的影响

在血液来源和血液制品安全隐患制约下,基因工程重组凝血八因子已开始替代天然八因子用以治疗血友病等病症,但目前高效表达重组八因子的方法在我国仍有技术瓶颈.该项目利用定点突变技术,通过PCR、质粒抽提、转染以及产物检测这四个步骤来研究重组凝血八因子在A和C结构域添加糖基化位点对八因子表达及活性的影响.结果表明,在重组八因子A结构域Bip结合位点外端增加糖基化位点能够促进八因子的细胞外分泌量,而在Bip结合位点内的突变则不但大大减少了八因子的胞外分泌也使八因子活性几乎全部丧失.与A结构域不同,在八因子C结构域增加糖基化位点的结果表明,无论在蛋白C结合区之内或之外增加糖基化位点不但不能大幅提高八因子的胞外分泌,也使八因子活性基本丧失.这些结果说明只有在A结构域外缘的非Bip结合区进行糖基化改造,才有可能提高八因子分泌并保持其活性.     

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.     

Thr but Asn of the N-glycosylation sites of PrP is indispensable for its misfolding

Prion protein (PrP) contains two N-linked glycosylation sites. It is unknown which amino acid substitution contributes most efficiently to the abolishment of N-linked glycosylations. To define the influence of amino acid substitution at the N-linked glycosylation sites on the conversion efficiency of mouse PrP, we tested each of all 19 amino acid substitutions at either one of the N-linked glycosylation sites (codon 180, 182, 196 or 198). The conversion efficiency of the mutagenized PrP was highly dependent on the newly introduced amino acid itself regardless of the absence of N-linked glycosylation in scrapie-infected mouse neuroblastoma cells. The majority of mutant PrP with substitutions at the Asn residues of the N-linked glycosylation sites were conversion-competent, whereas most mutant PrP with substitutions at the Thr residues were conversion-incompetent. These findings emphasize that the Asn residues of the N-linked glycosylation sites are replaceable to abolish N-linked glycosylations without directly affecting the protein function.     

One-pipeline approach achieving glycoprotein identification and obtaining intact glycopeptide information by tandem mass spectrometry

A novel one-pipeline approach is reported, which can demonstrate glycoprotein identification and obtain intact glycosylation information after glycopeptide-level enrichment, without de-glycosylation. The proposed workflow has two enrichment steps plus two proteolytic processes: enriched glycoproteins were digested to peptides by Lys-C, and then enriched again and secondly digested by trypsin. In the resulting mixture, with a reasonable complexity, intact glycopeptides could be preserved and utilized informatively for glycosylation analysis, and non-glycopeptides for protein identification. In both standard protein mixture tests and real sample analysis, the resulting glycopeptides and non-glycopeptides were proved to play their expected roles, thus more confident protein glycosylation information was obtained.     

Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.     

Comparing the use of 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl and 2,4,6-trimethoxybenzyl as N-H protecting groups for p-tolyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-D-glucosides

A hurdle in glycosylation reactions of 2-acetamido glycosyl donors is the formation of a stable and unreactive oxazoline that decreases the yield of these reactions significantly. As an effort to prevent oxazoline formation during glycosylation reactions, we protected the N-H of the acetamido group within a 2-acetamido-2-deoxy-1-thio-β-D-glucoside with one of four different protecting groups. These groups were either 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl or 2,4,6-trimethoxybenzyl. The resulting N-alkylacetamides were then used in glycosylation reactions with ethanol as a model acceptor. We observed that the ethyl glycosides obtained in each case were obtained with exclusive β-selectivity without the formation of oxazoline sideproducts. The resulting products were then used to screen conditions for protecting group removal.     

人朊蛋白不同N-糖基化修饰突变体的构建及其在哺乳动物细胞中表达

构建并表达人朊蛋白N-糖基化修饰位点突变的真核表达载体,有助于进一步研究朊蛋白N-糖基化修饰的生物学功能。定点突变野生型人朊蛋白基因PRNP,将获得的突变体亚克隆至真核表达载体pcDNA3.1中,并在人宫颈癌细胞株HeLa中瞬时表达各种朊蛋白糖基化修饰位点突变体,利用免疫印迹和糖苷酶消化等糖蛋白分析方法鉴定表达产物的糖基化形式。经Western blot鉴定,野生型和突变型朊蛋白表达产物出现不同形式的泳动特征,分别出现特异性糖基化修饰的多个条带,单糖基化修饰的两条条带和无糖基化修饰的一条条带。经PNGase F糖苷酶消化,野生型和糖基化单点突变型表达产物均能被糖苷酶消化,其分子量下移,去糖基化突变型表达产物的分子条带位置不变。通过突变野生型人朊蛋白基因PRNP的N-糖基化修饰位点,获得单糖基化修饰和去N-糖基化修饰的6种人朊蛋白突变体,并能够在HeLa细胞株中瞬时表达单糖基化修饰和去N-糖基化修饰朊蛋白,为进一步研究朊蛋白的相关功能建立良好基础。     

Comparing the use of 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl and 2,4,6-trimethoxybenzyl as N–H protecting groups for p-tolyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-d-glucosides

A hurdle in glycosylation reactions of 2-acetamido glycosyl donors is the formation of a stable and unreactive oxazoline that decreases the yield of these reactions significantly. As an effort to prevent oxazoline formation during glycosylation reactions, we protected the N–H of the acetamido group within a 2-acetamido-2-deoxy-1-thio-β-d-glucoside with one of four different protecting groups. These groups were either 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl or 2,4,6-trimethoxybenzyl. The resulting N-alkylacetamides were then used in glycosylation reactions with ethanol as a model acceptor. We observed that the ethyl glycosides obtained in each case were obtained with exclusive β-selectivity without the formation of oxazoline sideproducts. The resulting products were then used to screen conditions for protecting group removal.     

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44⁺/ESA⁺/CD24⁻ markers, Gb3 activates c-Src/β-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/β-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.