Role Of N-linked Glycosylation Research Articles

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.

Functional analysis of individual oligosaccharide chains of Sendai virus hemagglutinin-neuraminidase protein.

The roles of N-linked glycosylation in the intracellular transport and biological activities of the Sendai virus hemagglutinin-neuraminidase (HN) protein were studied. The protein contains four potential N-glycosylation sites: N77, N448, N499, and N511. By site-directed mutagenesis of these positions, the mature protein contained three N-linked oligosaccharides attached to N77, N499, and N511. The role of each added oligosaccharide in the structure and functions of the protein was identified by characterization of surface expression, hemadsorption, and neuraminidase activities of the corresponding mutant proteins. Elimination of the sites of N499 and N511 had the most detrimental effect, decreasing surface expression and hemadsorption. Elimination of the sites of N77 and N448 had similar but weaker effects. Mutants missing the sites of N499 and N511 were not able to induce syncytia formation in cells expressing mutant HN proteins and the fusion protein. Therefore, the N-linked oligosaccharides attached to N499 and N511 were important for intracellular transport and for the fusion promotion.

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.

Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein.

The roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking g1 and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.

Investigation of the role of N-linked glycosylation of the human Thromboxane A2 receptor

Investigation of the role of N-linked glycosylation of the human Thromboxane A2 receptor

Role of N-Linked Glycosylation on the Function and Expression of the Human Secretin Receptor

Role of N-Linked Glycosylation on the Function and Expression of the Human Secretin Receptor

Role of N-linked glycosylation on the function and expression of the human secretin receptor.

Secretin is a 27-amino acid long peptide hormone that regulates pancreatic water, bicarbonate, enzymes, and potassium ion secretion. The human secretin receptor (hSR) is a glycoprotein consisting of 440 amino acids, of which there are 5 putative N-linked glycosylation sites at positions Asn72, Asn100, Asn106, Asn128 (N-terminal ectodomain), and Asn291 (second exoloop). Through functional analysis of the hSR-transfected cells cultured in the presence of various glycosylation inhibitors, it was found that tunicamycin and castanospermine were able to significantly reduce the secretin-stimulated cAMP response. On the other hand, the effects of other inhibitors, swainsonine and deoxymannojirimycin, were much lower, suggesting that the high mannose-type carbohydrate side-chain is essential to the expression of a fully functional hSR. The role of individual N-linked glycosylation sites was studied by mutation analysis (Asn to Leu or Ser to Ala) coupled to measurements of cAMP accumulation and extracellular acidification rate. The ED50 values of the wild-type receptor in these two assay systems were 0.25 and 0.11 nM, respectively, and mutation at position 100, 106, or 291 did not affect either the ED50 values or the maximal responses in the two assays. However, the Asn72Leu and Ser74Ala mutations reduced the maximal responses and increased the ED50 values in both assays, suggesting that this site is a true glycosylation signal. This hypothesis was further supported by competitive binding studies, the same mutants were found to be defective in binding with [125I]secretin. To evaluate whether the change in receptor function of the mutants is caused by the change in the process of presenting the receptor to the cell surface, the mutants and the wild-type receptor were tagged with a c-Myc epitope at the C-termini. Using an anti-c-Myc monoclonal antibody and confocal microscopy, all of the mutant receptors were found to be expressed and delivered to the plasma membrane.

Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of [3H]NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10. 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0. 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0. 04)x10(8) M-1.min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means±S.D.) of [3H]NBMPR to intact yeast (Kd 1.2±0.2 nM; Bmax 5.0±0.5 pmol/mg of protein) and membranes (Kd 0.7±0.2 nM; Bmax 6.5±1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130±10 and 380±20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10.5±1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14±0.02 and 0.36±0.05 min-1 for koff, and (1.2±0.1)×108 and (0.40±0.04)×108 M-1·min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

The role of N-linked glycosylation in the secretion of hepatitis B virus.

Hepatitis B virus (HBV) is the human member of the hepadnaviradae family of viruses which chronically infects over 300 million people worldwide. Infection by HBV can lead to both acute and chronic liver disease. For those unfortunate to become chronic carriers of HBV, there are lifelong complication associated with the virus. Indeed up to 40% of chronic carriers may eventually develop hepatocellular carcinoma (1). As there is such a large pool of chronic carriers, it can be assumed that a vast number of the worlds population die from complications that arise from infection with HBV.

Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion.

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 alpha-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that alpha-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. alpha-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50=50 degrees C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 degrees C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 alpha-amylase in both its wild-type and mutant forms.

An N-Linked Glycosylation Motif from the Noncleaving Luteinizing Hormone Receptor Substituted for the Homologous Region (Gly367 to Glu369) of the Thyrotropin Receptor Prevents Cleavage at Its Second, Downstream Site

The thyrotropin receptor (TSHR) exists in two forms (single polypeptide and two subunits), whereas the lutropin/chorionic gonadotropin receptor (LH/CGR) is a single chain. Recent data suggest that the TSHR cleaves at two sites. We mutagenized selected chimeric TSH-LH/CGR to localize the cleavage sites in the TSHR. All 23 receptors mutated in the estimated vicinity of the upstream site cleaved into two subunits as determined by 125I-TSH cross-linking to intact cells. In contrast, in a series of mutations homologous to the noncleaving LH/CGR, the downstream TSHR cleavage site localized to three amino acids (GQE367-369). Remarkably, group substitution of these residues, but not substitution of individual residues, abolished cleavage. Moreover, the mutation that prevented cleavage (GQE367-369NET) transposed a motif (NET291-293) that is glycosylated in the LH/CGR. TSHR cleavage or noncleavage after substitution of GQE367-369 with other triplets (AAA, NQE, and NQT) was consistent with a role for N-linked glycosylation at this site. In summary, our data (i) support the concept that the TSHR cleaves at two sites, (ii) relate TSHR residues GQE367-369 to cleavage at the second, downstream site, and (iii) suggest that cleavage or noncleavage at site two is related to N-linked glycosylation. These findings provide new insight into the evolutionary divergence of two closely related receptors.

Role of individual N-linked oligosaccharide chains and different regions of bovine respiratory syncytial virus fusion protein in cell surface transport.

The role of N-linked glycosylation and various domains of the fusion (F) protein of bovine respiratory syncytial virus strain A51908 during transport to the cell surface were determined by mutating the four glycosylation sites and by constructing various truncation mutants. The cell surface expression of various mutant proteins was detected by surface immunofluorescence and cell surface biotinylation assay. We found that the glycosylation at positions Gly1, Gly2, Gly3, and a truncation mutant with a cytoplasmic domain deletion play little or no role in transport to cell surface. However, glycosylation at position Gly4, and the truncation mutants having deletion in the signal peptide, the F2 + signal peptide domain, and the membrane anchor domain, had impaired transport to the cell surface. This indicated the importance of these glycosylation sites and domains in facilitating the transport of the F protein to the cell surface.

Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion.

The role of N-linked glycosylation and glycan trimming in the function of glycoproteins remains a central question in biology. Hepatitis B virus specifies three glycoproteins (L, M, and S) that are derived from alternate translation of the same ORF. All three glycoproteins contain a common N-glycosylation site in the S domain while M possesses an additional N-glycosylation site at its amino terminus. In the presence of N-butyl-deoxnojirimycin (an inhibitor of alpha-glucosidase) virions and the M protein are surprisingly retained. Preliminary evidence suggests that the retained M protein is hyperglucosylated and localized to lysosomal vesicles. In contrast, the S and L proteins are secreted, and their glycosylation state is unaffected by the presence of the inhibitor. Site-directed mutagenesis provides evidence that virion secretion requires the glycosylation sequon in the pre-S2 domain of M. This highlights the potential role of the M protein oligosaccharide as a therapeutic target.

Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Role of N-linked glycosylation in expression of E-selectin on human endothelial cells.

E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.

Individual Roles of N-Linked Oligosaccharide Chains in Intracellular Transport of the Paramyxovirus SV5 Fusion Protein

The role of N-linked glycosylation in the assembly, intracellular transport, and fusion activity of the paramyxovirus SV5 fusion (F) protein was examined. Each of the six potential glycosylation sites in the F protein was individually removed by oligonucleotide-directed mutagenesis on a cDNA clone encoding the SV5 F protein. When the mutant F proteins were expressed in eukaryotic cells using the vaccinia virus-T7 transient expression system they all had a significant change in gel mobility, indicating that all six sites in the F protein are used for the addition of N-linked oligosaccharides. All of the mutant F proteins could form a homooligomer. Removal of individual carbohydrate chains from the F 2 subunit had little effect on the surface expression of the F protein. However, removal of individual carbohydrate chains from the F 1 subunit had deleterious effects, which ranged from a partial delay in intracellular transport and decreased stability of the protein to severe transport delays and acute instability of the F protein.

Tunicamycin Inhibits the Expression of Functional Thrombin Receptors on Human T-Lymphoblastoid Cells

N-linked glycosylation plays an important role in intracellular trafficking and the surface expression of many membrane-associated proteins. In the present report we investigated the effect of tunicamycin, a specific inhibitor of N-linked glycosylation, on the expression and function of the thrombin receptor on human T-lymphoblastoid cells. We found that tunicamycin selectively inhibited thrombin-induced Ca2+ mobilization in a dose-dependent manner while the same response triggered by anti-CDS antibody was unchanged in these cells. Surface expression of the thrombin receptor, as assessed by immunofluorescence staining using two different antibodies, was strongly decreased in the presence of tunicamycin. These findings indicate a role for N-linked glycosylation in the surface expression of the thrombin receptor in T lymphoid cells.

Role of N-linked glycosylation in rat renal Na/Pi-cotransport.

Our laboratory recently identified a sodium-dependent transport system for phosphate from rat kidney cortex (NaPi-2; Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5979-5983). In the present study we have investigated whether or not this cotransporter is glycosylated and the role of N-glycosylation in determining its function. Glycosidase digestion of the NaPi-2 protein from rat brush border membranes, in vitro translation studies, or oocyte expression of the NaPi-2 cRNA indicate that the mature protein is glycosylated. Glycosidase treatment reduces the size of the protein from approximately 70-110 kDa to approximately 60-65 kDa. We therefore used site-directed mutagenesis to identify which of the putative consensus sites for N-linked glycosylation are utilized in the mature NaPi-2 protein. Altering the nucleotide sequences encoding both of the Asn-298 and Asn-328 residues to Gln produced mutants that are completely devoid of glycosylation, whereas mutants in which each of these sites were mutated separately are glycosylated when expressed in oocytes. These results suggest that both of these sites are modified by N-linked glycosylation in the mature protein. Surface expression of glycosylated and unglycosylated NaPi-2-related proteins was documented by biotinylation experiments. In contrast to the wild-type (fully glycosylated) transporter, immunocytochemistry provides evidence for a partial intracellular localization of mutant unglycosylated cotransporters. Na/Pi cotransport was studied in oocytes expressing wild-type or mutagenized NaPi-2 proteins using tracer or electrophysiological techniques. Although the transport rates are lower (by a factor of 2-3) after expression of the unglycosylated NaPi-2 protein, the Pi transport characteristics (pH dependence, apparent affinity for Pi or Na+) are similar in oocytes expressing either wild-type or glycosylation-deficient proteins.

Role of N-linked Glycosylation in the Activity of the Friend Murine Leukemia Virus SU Protein Receptor-Binding Domain

The 243 N-terminal residues of Friend Murine Leukemia Virus envelope glycoprotein (SU) fold into a structurally and functionally autonomous domain which contains the determinants for binding to the ecotropic virus receptor. The two N-linked glycosylation sites present in this N-terminal portion of the viral SU were removed by site-directed mutagenesis without disturbing its biosynthesis and incorporation into infectious virions. A truncated version of the mutant protein which included only the N-terminal domain was poorly transported but still able to interact with the receptor. Interference assays Indicated that the interaction between the mutated protein and the virus receptor was weaker. We conclude that the elimination of N-linked oligosaccharide chains in the envelope N-terminal domain do not prevent receptor interaction but results in subtle conformational changes that may alter recognition and binding.