Activated protein C N-linked glycans modulate cytoprotective signaling function on endothelial cells

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporine-induced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma β-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.     

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL2, and HDL3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL2.     

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.     

The structural role of N-linked glycans on human glypican-1

Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was reduced and that the protein did not accumulate inside the cells. Heparan sulfate substitution of N-glycosylation mutant N116Q was similar to wild-type glypican-1 while the N79Q mutant and also the double mutant N79Q,N116Q were mostly secreted as high-molecular-weight heparan sulfate proteoglycan. N-Glycosylation mutants and N-deglycosylated glypican-1 had far-UV circular dichroism and fluorescence emission spectra that were highly similar to those of N-glycosylated glypican-1. A single unfolding transition at high concentrations of urea was found for both N-deglycosylated glypican-1 and glypican-1 in which the N-glycosylation sites had been removed by mutagenesis when chemical denaturation was monitored by circular dichroism and fluorescence emission spectroscopy. In summary, we have found that the potential N-glycosylation sites in glypican-1 are invariably occupied and that the N-linked glycans on glypican-1 affect protein expression and heparan sulfate substitution but that correct folding can be obtained in the absence of N-linked glycans.     

Post-translational processing events in the secretion pathway of human protein C, a complex vitamin K-dependent antithrombotic factor.

Human protein C (HPC) undergoes several post-translational modifications, including gamma-carboxylation, N-linked glycosylation, and internal proteolytic processing. We have utilized a recombinant human kidney cell line (293) secreting correctly modified HPC (rHPC) to study the processing reactions for the modification of this complex protein. gamma-Carboxylation was shown to proceed via a vitamin K-dependent pathway and was required for both efficient secretion and anticoagulant activity. rHPC was rapidly secreted following the addition of vitamin K to depleted cells, and secretion was not inhibited by cyclohexamide indicating that non gamma-carboxylated rHPC accumulates as an intracellular releasable pool. However, in cells grown in the presence of vitamin K, the majority of intracellular rHPC was gamma-carboxylated, suggesting that this post-translational modification is not rate limiting for secretion under conditions optimal for vitamin K-dependent carboxylation. Nonglycosylated rHPC was found to be secreted inefficiently, and processing of the N-linked core in the endoplasmic reticulum, but not in the Golgi, was required for secretion. Further, the intracellular rHPC present in vitamin K-supplemented cells was core glycosylated, but not processed past the high mannose step. gamma-Carboxylation occurred after core glycosylation, indicating that this modification is not cotranslational. Further, glycosylation and gamma-carboxylation were not coupled and did not need to proceed sequentially. Proteolytic processing of the internal KR dipeptide was found to occur late in the secretion pathway, and the cleavage was calcium-dependent. The secretion rate of rHPC was also calcium-dependent but was independent of the calcium effect on internal KR dipeptide removal, indicating that cleavage is not required for efficient secretion. Our results define the sequence of processing events, the subcellular localization of the processing reactions, and the rate-limiting steps in the secretion pathway for this complex protein.     

Translation rate of human tyrosinase determines its N-linked glycosylation level

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.     

Essential role of dengue virus envelope protein N glycosylation at asparagine-67 during viral propagation

Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.     

Incorporation of beta-fluoroasparagine into peptides prevents N-linked glycosylation. In vitro studies with synthetic fluoropeptides

Previously, we reported that incorporation of threo-beta-fluoroasparagine into cellular protein inhibits N-linked glycosylation. We now show that short synthetic peptides which contain N-acetyl-threo-beta-fluoroasparagine fail to undergo glycosylation in a cell-free system except at extremely high substrate concentrations. An N-benzoyl-threo-beta-fluoroasparagine-containing peptide has a 100-fold lower Vmax/Km than the analogous N-benzoyl-asparagine-containing peptide. Substitution of a fluorine for a hydrogen on the beta-carbon of asparagine weakens the ability of the peptide to bind the oligosaccharyltransferase. A 100-fold excess of acetyl-threo-beta-fluoroasparaginyl-leucyl-threonine methylamide over acetyl-asparaginyl-leucyl-threonine methylamide inhibited glycosylation of the latter peptide by less than 10%. Both threo-beta-fluoroasparagine and erythro-beta-fluoroasparagine-containing peptides are glycosylated at the same rate. Glycofluoropeptides generated from beta-fluoroasparagine-containing peptides were N-glycosylated. These cell-free studies with synthetic fluoropeptides suggest that incorporation of beta-fluoroasparagine into cellular protein inhibits N-linked glycosylation by rendering protein substrates ineffective for glycosylation. In the course of this work, we also demonstrate that the N-linked glycosylating enzyme acts only on L-asparagine-containing peptides and not on D-asparagine peptides.     

Mutational analysis of the role of N-glycosylation in alpha-factor receptor function

The alpha-factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha-factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.     

Site-specific processing of the N-linked oligosaccharides of the human chorionic gonadotropin alpha subunit

Two forms of the gonadotropin alpha subunit are synthesized in placenta and in human chorionic gonadotropin (hCG)-producing tumors: an uncombined (monomer) form and a combined (dimer) form. These forms show differences in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The slower migration of the monomeric form on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been attributed to a different glycosylation pattern. Previous studies demonstrated different roles of each of the two alpha N-linked glycosylation sites (Asn-52 and Asn-78) in secretion of the uncombined subunit and the biologic activity of hCG dimer. To assess the influence of formation of dimer on the processing pattern at the individual sites, we characterized the N-linked oligosaccharides of monomer and dimer forms of recombinant human choriogonadotropin alpha subunit. Two approaches were employed. First, site-directed mutagenesis was used to alter the two N-linked oligosaccharide attachment sites, thus allowing the expression of alpha subunits containing only one glycosylation site. Second, tryptic glycopeptides of the wild-type subunits were examined. Concanavalin A (ConA) binding and sialic acid content indicated that the oligosaccharides at each glycosylation site of the uncombined alpha subunit are processed differently. Oligosaccharides present at Asn-52 are almost exclusively ConA-unbound and contain three sialic acid residues. The majority of Asn-78-linked oligosaccharides are ConA-bound and disialylated. Both sites are processed independently because no significant differences were observed between the oligosaccharides at the same sites in wild-type and mutant monomeric alpha subunits. By contrast, the majority of the oligosaccharides at both glycosylation sites of the dimer alpha are bound to ConA. Thus, combination primarily affects the processing pattern of the Asn-52-linked species. Because glycosylation at this site is essential for hCG assembly and signal transduction, these data imply a critical link between the site-specific processing and hormone function.     

Inhibition of N-linked glycosylation affects organic cation transport across the brush border membrane of opossum kidney (OK) cells.

Many organic cations are transported across the apical membrane of the proximal tubule by specific saturable mechanisms. The goal of this study was to determine if the transporter for tetraethylammonium (TEA) in the brush border membrane of an established opossum kidney (OK) cell line is glycosylated and to elucidate the function of this glycosylation. The uptake of TEA was determined in OK cell monolayers treated with tunicamycin (TM), a compound that prevents synthesis of the core oligosaccharide precursor molecules. TM exposure significantly decreased the incorporation of [3H]mannose in OK cell proteins and significantly reduced TEA uptake in a time and a concentration dependent manner. No effect of TM exposure on cellular protein synthesis, DNA content, cell viability, or on [3H]proline uptake was observed. The transport of TEA in control cells was characterized by a Km of 26.9 +/- 16.4 microM and a Vmax of 378 +/- 39 pmol/mg of protein/min. TM treatment (1 microgram/ml for 21 h) significantly increased the Km by over 4-fold to 111.5 +/- 18.4 microM while not affecting the Vmax. The apparent KI values of other organic cations known to interact with this transport system were also significantly increased by TM exposure. Estimated KI values of N1-methylnicotinamide, cimetidine, and mepiperphenidol increased by 6-fold, 4-fold, and 2-fold, respectively, after exposure of OK cells to TM. An increased KI for protons was also observed. Additional inhibitors of the N-linked glycosylation pathway, castanospermine, deoxynojirimycin, and deoxymannojirimycin significantly decreased TEA transport, whereas swainsonine had no effect. Our results suggest that the organic cation transporter is glycosylated. The N-linked oligosaccharide side chain appears to be of the hybrid type, and it either directly or indirectly affects the binding site of the transporter for both organic cations and protons. This is the first report describing the importance of glycosylation in the function of the organic cation transporter in the apical membrane of OK cells.     

Role of N-linked glycosylation in the secretion and activity of endothelial lipase

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K(m) and increased apparent V(max). Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V(max). Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.     

Critical role of Dengue Virus NS1 protein in viral replication

Dengue virus (DENV) nonstructural protein 1 (NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites (Asn-130 and Asn-207) and 12 conserved cysteine (Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites (Cys-4, Cys-55, Cys-291) and a C-terminal deletion (ΔC) mutant significantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type (WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulfide bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.     

The G82S polymorphism promotes glycosylation of the receptor for advanced glycation end products (RAGE) at asparagine 81: comparison of wild-type rage with the G82S polymorphic variant

Interaction between the receptor for advanced glycation end products (RAGE) and its ligands amplifies the proinflammatory response. N-Linked glycosylation of RAGE plays an important role in the regulation of ligand binding. Two potential sites for N-linked glycosylation, at Asn(25) and Asn(81), are implicated, one of which is potentially influenced by a naturally occurring polymorphism that substitutes Gly(82) with Ser. This G82S polymorphic RAGE variant displays increased ligand binding and downstream signaling. We hypothesized that the G82S polymorphism affects RAGE glycosylation and thereby affects ligand binding. WT or various mutant forms of RAGE protein, including N25Q, N81Q, N25Q/G82S, and N25Q/N81Q, were produced by transfecting HEK293 cells. The glycosylation patterns of expressed proteins were compared. Enzymatic deglycosylation showed that WT RAGE and the G82S polymorphic variant are glycosylated to the same extent. Our data also revealed N-linked glycosylation of N25Q and N81Q mutants, suggesting that both Asn(25) and Asn(81) can be utilized for N-linked glycosylation. Using mass spectrometry analysis, we found that Asn(81) may or may not be glycosylated in WT RAGE, whereas in G82S RAGE, Asn(81) is always glycosylated. Furthermore, RAGE binding to S100B ligand is affected by Asn(81) glycosylation, with consequences for NF-κB activation. Therefore, the G82S polymorphism promotes N-linked glycosylation of Asn(81), which has implications for the structure of the ligand binding region of RAGE and might explain the enhanced function associated with the G82S polymorphic RAGE variant.     

O-linked glycosylation at threonine 27 protects the copper transporter hCTR1 from proteolytic cleavage in mammalian cells

The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wild-type hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.     

Functional properties of carbohydrate-depleted tissue plasminogen activator

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.     

Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.     

Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function

The relaxin receptor, RXFP1, is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346, which are highly conserved across species. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors, although its role in modulating receptor function differs. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at individual or combinations of double and triple sites had little effect on relaxin binding. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling.     

Targeted mass spectrometric approach for biomarker discovery and validation with nonglycosylated tryptic peptides from N-linked glycoproteins in human plasma

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.     

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M. and Kobata, A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.