scholarly journals N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

1999 ◽  
Vol 339 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Benoit DESLAURIERS ◽  
Cecilia PONCE ◽  
Colette LOMBARD ◽  
Renée LARGUIER ◽  
Jean-Claude BONNAFOUS ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Cellular Localization ◽  
Inositol Phosphate ◽  
Glycosylation Site ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Pharmacological Properties ◽  
Peptide Epitope ◽  
Glycosylation Sites

The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn → Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.

scholarly journals Requirement of N-glycosylation of the prostaglandin E2 receptor EP3β for correct sorting to the plasma membrane but not for correct folding

2000 ◽  
Vol 350 (3) ◽  
pp. 839-847 ◽  
Author(s):  
Ulrike BÖER ◽  
Frank NEUSCHÄFER-RUBE ◽  
Ulrike MÖLLER ◽  
Gerhard P. PÜSCHEL
Keyword(s):  
Plasma Membrane ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Confocal Laser ◽  
Hek 293 ◽  
Consensus Sequences ◽  
Glycosylation Sites ◽  
293 Cells ◽  
Prostaglandin E2 Receptor

Eight heptahelical receptors have been characterized for prostaglandin (PG) D2, PGE2, PGF2α, prostacyclin and thromboxane A2. They share a sequence identity of 40%. All of them have potential N-glycosylation sites. The current study analysed the role of the two N-glycosylation sites in the rat EP3β-subtype PGE2 receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutagenesis and receptors expressed in HEK-293 cells. Both potential N-glycosylation sites were used. Their joint elimination resulted in the synthesis of a receptor protein with full binding competence, biological activity and no reduction of affinity; however, the half-life of the non-glycosylated receptor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosylated receptor was correctly inserted into the plasma membrane to a much larger extent than the non-glycosylated receptor, which tended to accumulate in the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycosylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3β receptor seems not to be necessary for correct folding of the receptor protein but for the efficient transport of the receptor protein to the plasma membrane. This contrasts with a previous finding which described a reduction of the affinity for PGE2 of the EP3α receptor by elimination of the distal glycosylation site when the receptor protein was expressed in insect cells.

Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

2008 ◽  
Vol 294 (4) ◽  
pp. G1052-G1059 ◽  
Author(s):  
Pijun Wang ◽  
Soichiro Hata ◽  
Yansen Xiao ◽  
John W. Murray ◽  
Allan W. Wolkoff
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Integral Membrane Protein ◽  
Glycosylation Site ◽  
Domain Model ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Glycosylation Sites

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

scholarly journals Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue

1997 ◽  
Vol 326 (1) ◽  
pp. 243-247 ◽  
Author(s):  
Gilles MILLAT ◽  
Roseline FROISSART ◽  
Irène MAIRE ◽  
Dominique BOZON
Keyword(s):  
Catalytic Activity ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Lysosomal Storage ◽  
Post Translational Modification ◽  
Cos Cells ◽  
Conserved Sequence ◽  
Glycosylation Sites

Iduronate sulphatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to evaluate the functional importance of each N-glycosylation site, and of the cysteine-84 residue. IDS mutant cDNAs, lacking one of the eight potential N-glycosylation sites, were expressed in COS cells. Although each of the potential sites was used, none of the eight glycosylation sites appeared to be essential for lysosomal targeting. Another important sulphatase co- or post-translational modification for generating catalytic activity involves the conversion of a cysteine residue surrounded by a conserved sequence C-X-P-S-R into a 2-amino-3-oxopropionic acid residue [Schmidt, Selmer, Ingendoh and von Figura (1995) Cell 82, 271–278]. This conserved cysteine, located at amino acid position 84 in IDS, was replaced either by an alanine (C84A) or by a threonine (C84T) using site-directed mutagenesis. C84A and C84T mutant cDNAs were expressed either in COS cells or in human lymphoblastoid cells deleted for the IDS gene. C84A had a drastic effect both for IDS processing and for catalytic activity. The C84T mutation produced a small amount of mature forms but also abolished enzyme activity, confirming that the cysteine residue at position 84 is required for IDS activity.

Effects of N224 glycosylation in Saccharomycopsis fibuligera R64 α-amylase on enzyme activity and stability

2021 ◽  
Vol 17 ◽  
Author(s):  
Yovin Sugijo ◽  
Tina Dewi Rosahdi ◽  
Fernita Puspasari ◽  
Wangsa Tirta Ismaya ◽  
Khomaini Hasan ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Evolutionary Relationship ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Saccharomycopsis Fibuligera ◽  
Glycosylation Sites ◽  
Starch Substrate ◽  
Relationship Of

Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains the two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. Objective: To test whether N224 plays a key role in enzyme activity and stability. Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activity of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

N-glycosylation of CRF receptor type 1 is important for its ligand-specific interaction

2001 ◽  
Vol 281 (5) ◽  
pp. E1015-E1021 ◽  
Author(s):  
Iman Q. Assil ◽  
Abdul B. Abou-Samra
Keyword(s):  
High Efficiency ◽  
Specific Interaction ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Receptor Type ◽  
Ligand Interaction ◽  
Glycosylation Sites ◽  
Low Levels ◽  
Camp Stimulation

The corticotropin-releasing factor (CRF) receptor type 1 (CRFR1) contains five potential N-glycosylation sites: N38, N45, N78, N90, and N98. Cells expressing CRFR1 were treated with tunicamycin to block receptor glycosylation. The nonglycosylated receptor did not bind the radioligand and had a decreased cAMP stimulation potency in response to CRF. To determine which of the polysaccharide chain(s) is/are involved in ligand interaction, the polysaccharide chains were deleted using site-directed mutagenesis of the glycosylation consensus, N-X-S/T. Two sets of mutations were performed for each glycosylation site: N to Q and S/T to A, respectively. The single mutants Q38, Q45, Q78, Q90, Q98, A40, A47, A80, A92, and A100 and the double mutants A40/A47 and A80/A100 were well expressed, bound CRF, sauvagine (SVG), and urotensin-I (UTS-I) with a normal affinity, and increased cAMP accumulation with a high efficiency. In contrast, the combined mutations A80/A92/A100, A40/A80/A92/A100, and A40/A47/A80/A92/A100 had low levels of expression, did not bind the radioligand, and had a decreased cAMP stimulation. These data indicate the requirement for three or more polysaccharide chains for normal CRFR1 function.

Identification of the glycosylation sites utilized on the V1a vasopressin receptor and assessment of their role in receptor signalling and expression

2001 ◽  
Vol 357 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Stuart R. HAWTIN ◽  
Andrew R. L. DAVIES ◽  
Glenn MATTHEWS ◽  
Mark WHEATLEY
Keyword(s):  
Cell Surface ◽  
Large Family ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Vasopressin Receptor ◽  
Receptor Signalling ◽  
Glycosylation Sites ◽  
And Function ◽  
Translation Systems ◽  
G Protein Coupled

Most of the large family of G-protein-coupled receptors (GPCRs) possess putative N-glycosylation sites within their N-termini. However, for the vast majority of GPCRs, it has not been determined which, if any, of the consensus glycosylation sites are actually utilized or what the functional ramifications are of modification by oligosaccharide. The occurrence and function of glycosylation of the V1a vasopressin receptor (V1aR) has been investigated in this study. Using a combination of translation systems that are either glycosylation-competent or do not support glycosylation, we established that of the four putative N-glycosylation sites at Asn14, Asn27, Asn198 and Asn333 only the first three sites are actually modified by carbohydrate. This was confirmed by disruption of consensus sites by site-directed mutagenesis, individually and in combination. The V1aR is not O-glycosylated. The functionality of a series of glycosylation-defective V1aR constructs was characterized after expression in HEK 293T cells. It was found that carbohydrate moieties are not required for the receptor to bind any of the four classes of ligand available, or for intracellular signalling. The glycosylation status of the V1aR did, however, regulate the level of total receptor expression and also the abundance of receptor at the cell surface. Furthermore, the nature of this regulation (increased or decreased expression) was dictated by the locus of the oligosaccharide modification. Modification of any one of the consensus sites alone, however, was sufficient for wild-type expression, indicating a redundancy within the glycosylation sites. A role for the carbohydrate in the correct folding or stabilization of the V1aR is indicated. Glycosylation is not required, however, for efficient trafficking of the receptor to the cell surface. This study establishes the functional importance of N-glycosylation of the V1aR.

scholarly journals Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site

2002 ◽  
Vol 70 (6) ◽  
pp. 2837-2845 ◽  
Author(s):  
Jason E. Comer ◽  
Mark A. Marshall ◽  
Vincent J. Blanch ◽  
Carolyn D. Deal ◽  
Peter Castric
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Epitope ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Epitope ◽  
B Cell Epitope ◽  
Terminal Amino ◽  
Carboxy Terminal

ABSTRACT Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.

scholarly journals Lecithin:cholesterol acyltransferase: role of N-linked glycosylation in enzyme function

1993 ◽  
Vol 294 (3) ◽  
pp. 879-884 ◽  
Author(s):  
K O ◽  
J S Hill ◽  
X Wang ◽  
R McLeod ◽  
P H Pritchard
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Lecithin:Cholesterol Acyltransferase ◽  
Mutant Proteins ◽  
Glycosylation Sites

Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is a glycoprotein which is responsible for the formation of cholesteryl ester in plasma. The carbohydrate content has been estimated to be approx. 25% of the total LCAT mass, and four potential N-linked glycosylation sites have been predicted at residues 20, 84, 272 and 384 of the LCAT protein sequence. In the present study, we have examined which of these sites are utilized and how the N-glycosylation affects the secretion and function of the enzyme. Site-directed mutagenesis was performed to eliminate the glycosylation consensus sequence at each of the four potential sites, and the mutant proteins were expressed in COS cells. The amount of each mutant LCAT secreted was similar to that of the wild-type enzyme but the molecular mass was decreased by 3-4 kDa. The specific activity of each mutant LCAT was significantly different from the wild-type; however, the magnitude and direction of the change depended on the glycosylation site mutagenized. Loss of carbohydrate at position 20, 84 or 272 resulted in a decrease in the specific activity of the mutant enzymes by 18%, 82%, and 62% respectively. In contrast, the mutant protein without glycosylation at position 384 displayed a 2-fold increase in enzyme activity. In addition, a quadruple mutant was constructed such that all four potential glycosylation sites were eliminated. The amount of the unglycosylated LCAT secreted into the culture medium was less than 10% of the wild-type level and the specific activity of this enzyme was decreased to 5% of that of the wild type. The results demonstrate that all four potential N-glycosylation sites in LCAT are used and the presence of carbohydrate at each site has diverse effects on the enzyme activity.

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