Changes in N-glycosylation of human stromal cells by telomerase expression

It was established that remarkable changes in the N-glycosylation are induced in immortalized cancer cells. Whether changes were induced in human stromal cells immortalized by transfection with the human telomerase catalytic subunit (hTert) cDNA was examined by lectin blot analysis. Morphological appearance and growth rate of the gene-transfected stromal cells were not changed significantly. However, lectin blot analysis of membrane glycoprotein samples showed that bindings of Ricinus communis agglutinin-I (RCA-I) and of leuko-agglutinating phytohemagglutinin to glycoprotein bands increase significantly in the gene-transfected cells. No lectin binding was observed when blotted filters were treated with diplococcal beta-1,4-galactosidase or N-glycanase prior to incubation with RCA-I. In contrast, no changes in Coomassie brilliant blue-staining and in binding of concanavalin A were obtained between the primary and gene-transfected stromal cells. These results indicate that the highly branched N-glycosylation with augmented galactosylation is induced in human stromal cells immortalized by the telomerase expression.     

Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII

N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.     

Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

ABSTRACT

The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.     

Functions of human bitter taste receptors depend on N-glycosylation

Human bitter taste receptors of the TAS2R gene family play a crucial role as warning sensors against the ingestion of toxic food compounds. Moreover, the genetically highly polymorphic hTAS2Rs recognize an enormous number of structurally diverse toxic and non-toxic bitter substances, and hence, may substantially influence our individual eating habits. Heterologous expression in mammalian cells is a useful tool to investigate interactions between these receptors and their agonists. However, many bitter taste receptors are poorly expressed at the cell surface of heterologous cells requiring the addition of plasma membrane export promoting epitopes to the native receptor proteins. Currently, nothing is known about amino acid motifs or other receptor-intrinsic features of TAS2Rs affecting plasma membrane association. In the present study, we analyzed the Asn-linked glycosylation of hTAS2Rs at a consensus sequence in the second extracellular loop, which is conserved among all 25 hTAS2Rs. Non-glycosylated receptors exhibit substantially lower cell surface localization and reduced association with the cellular chaperone calnexin. As the auxiliary factors receptor transporting proteins 3 and 4 are able to restore the function of non-glycosylated hTAS2R16 partially, we conclude that glycosylation is important for receptor maturation but not for its function per se .     

Subtle evolutionary changes in the distribution of N-glycosylation sequons in the HIV-1 envelope glycoprotein 120

Many viruses are known to undergo rapid evolutionary changes under selective pressures. The HIV-1 envelope glycoprotein 120 (gp120) shows extreme selection for NXS/T sequons, the potential sites of N-glycosylation. Although the average number of sequons in gp120 appears to be relatively stable in the recent past, even slight changes in the distribution of sequons may potentially play crucial roles in protein interaction and viral infection. This study tracked the prevalence and distribution of NXS/T sequons in gp120 over a period of 29 years (from 1981 to 2009). The gp120 showed location specific distribution of sequons with higher density in the outer domain of the molecule. The NXT sequon density decreased in the outer domain (despite the increase in the sequon specific amino acid threonine), but increased in the inner domain. By contrast, the NXS sequon density increased specifically in the outer domain. Related changes were also seen in the distribution probabilities of sequons in two domains. The results indicate that the gp120, chiefly in subtype B, is redistributing NXS/T sequons within the molecule with specific selection for NXS sequons. The subtle evolution of sequons in gp120 may have implications in viral resistance and infection.     

Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.     

The role of N-glycosylation sites in the activity,stability, and expression of the recombinant elastase expressed by Pichia pastoris

The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70 °C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE.     

文化的起源——动物文化的普遍性与人类文化的独特性

在很长一段时间里文化被认为是人类文明的产物.该文提供了许多关于动物文化现象的证据,并且提供了动物创新和社会学习能力的观察与实证研究的结果,用以说明文化在动物中存在的可能性.然而,人类文化与动物文化存在着明显差异,造成这种结果的原因可能主要是两者认知能力的差异:人类特殊的认知模式--文化认知造就了人类复杂的文化体系.     

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60 kDa accompanied by a broad band of 60–90 kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53 kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn¹⁶¹ and Asn²⁴¹ on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.     

Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells

Mutations in the gene coding for a human ABC transporter protein, ABCC6 (MRP6), are responsible for the development of pseudoxanthoma elasticum. Here, we demonstrate that human ABCC6, when expressed by retroviral transduction in polarized mammalian (MDCKII) cells, is exclusively localized to the basolateral membrane. The human ABCC6 in MDCKII cells was found to be glycosylated, in contrast to the underglycosylated form of the protein, as expressed in Sf9 cells. In order to localize the major glycosylation site(s) in ABCC6, we applied limited proteolysis on the fully glycosylated and underglycosylated forms, followed by immunodetection with region-specific antibodies for ABCC6. Our results indicate that Asn15, which is located in the extracellular N-terminal region of human ABCC6, is the only N-glycosylation site in this protein. The polarized mammalian expression system characterized here provides a useful tool for further examination of routing, glycosylation, and function of the normal and pathological variants of human ABCC6.     

Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives

We report seven new members of the superfamily of human G protein-coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR100, GPR119, GPR120, GPR135, GPR136, GPR141, and GPR142. We also report 16 orthologues of these receptors in mouse, rat, fugu (pufferfish) and zebrafish. Phylogenetic analysis shows that these are additional members of the family of rhodopsin-type GPCRs. GPR100 shows similarity with the orphan receptor SALPR. Remarkably, the other receptors do not have any close relative among other known human rhodopsin-like GPCRs. Most of these orphan receptors are highly conserved through several vertebrate species and are present in single copies. Analysis of expressed sequence tag (EST) sequences indicated individual expression patterns, such as for GPR135, which was found in a wide variety of tissues including eye, brain, cervix, stomach and testis. Several ESTs for GPR141 were found in marrow and cancer cells, while the other receptors seem to have more restricted expression patterns.     

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.     

真菌N-糖基化系统在异源表达中的研究进展

在近20年来,越来越多的真菌被开发成异源蛋白表达宿主,用来生产各种药用蛋白和酶类。随着对真菌异源表达系统的研究,人们也渐渐意识到真菌N-糖基化系统与高等动物的N-糖基化系统有着明显的区别,这也成为真菌生产高等动物源性糖蛋白的一个技术瓶颈。本文综述了真菌在异源表达糖蛋白工程中其N-糖基化系统的研究进展。包括N-糖基的检测技术和改造策略,并重点介绍了真菌N-糖基化系统与高等动物的N-糖基化系统的差异,以期为日后真菌N-糖基化系统动物源化甚至人源化改造提供参考。     

The evolution of BIR domain and its containing proteins

BIR domain and its containing proteins play critical roles in cell apoptosis and cell division. Here several lines of novelty were revealed based on a comprehensive evolutionary analysis of BIR domains in 11 representative organisms. First, the type II BIR domains in Survivin and Bruce showed more conservation compared with the type I BIR domains in the inhibitors of apoptosis proteins (IAPs). Second, cIAP was derived from a XIAP duplicate and emerged just after the divergence of invertebrates and vertebrates. Third, the three BIR domains of NAIP displayed significantly elevated evolutionary rates compared with the BIR domains in other IAPs.     

Site-directed removal of N-glycosylation sites in human gastric lipase.

Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chains account for about 15% of the molecular mass of the native HGL. Four potential N-glycosylation consensus sites (Asn15, 80, 252 and 308) can be identified from the HGL amino acid sequence. We studied the functional role of the individual N-linked oligosaccharide chains by removing one by one all the N-glycosylation sites, via Ala residue replacement by site-directed mutagenesis of Ser and Thr residues from the consensus sequences Asn-X-Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculovirus/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 252 was found to have no significant influence on the enzymatic activity measured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombinant HGL (r-HGL), measured on short- and long-chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r-HGL markedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r-HGL and the completely deglycosylated r-HGL (four-site mutant) were found to be identical when recording the changes with time of the surface pressure either at the air-water interface or in the presence of an egg phosphatidylcholine (PtdCho) monomolecular film spread at various initial surface pressures. This indicates that both recombinant HGLs are identical, as far as recognition of phospholipid film and adsorption on PtdCho are concerned. The N-glycosylation of HGL may contribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r-HGL is increased.     

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。     

Glycosyltransferases and oligosaccharyltransferases in Archaea: putative components of the N-glycosylation pathway in the third domain of life

The ability of Eukarya, Bacteria and Archaea to perform N -glycosylation underlies the importance and possible antiquity of this post-translational protein modification. However, in contrast to the relatively well-studied eukaryal and bacterial pathways, the archaeal N -glycosylation process is less understood. To remedy this disparity, the following study has examined 56 available archaeal genomes with the aim of identifying glycosyltransferases and oligosaccharyltransferases, including those putatively catalyzing this post-translational processing event. This analysis reveals that while oligosaccharyltransferases, central components of the N -glycosylation pathway, are found across the range of archaeal phenotypes, the N -glycosylation machinery of hyperthermophilic Archaea may well rely on fewer components than do the parallel systems of nonhyperthermophilic Archaea. Moreover, genes encoding predicted glycosyltransferases of hyperthermophilic Archaea tend to be far more scattered within the genome than is the case with nonhyperthermophilic species, where putative glycosyltransferase genes are often clustered around identified oligosaccharyltransferase-encoding sequences.     

The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.