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.

Crucial Role of Complement Receptor Type 1 On the Accumulation of Complement Component 3 On Erythrocytes in Patients with PNH Treated with Eculizumab

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 988-988
Author(s):  
Masaki Yamamoto ◽  
Jun-ichi Nishimura ◽  
Makiko Oosato ◽  
Satoru Hayashi ◽  
Yoshiko Murakami ◽  
...  
Keyword(s):  
Crucial Role ◽  
Clinical Symptoms ◽  
Complement Component ◽  
Receptor Type ◽  
Complement Receptor ◽  
Expression Levels ◽  
Complement Receptor Type ◽  
Low Levels ◽  
Kinetics Of

Abstract Abstract 988 Paroxysmal nocturnal hemoglobinuria (PNH) is a hematologic disorder characterized by the clonal expansion of hematopoietic stem cells that are incapable of glycosylphosphatidylinositol (GPI)-anchor biosynthesis, due to an acquired somatic mutation in the PIGA gene. Affected progeny cells are deficient in all GPI-anchored surface proteins, including the complement regulators CD55 and CD59. Deficiency of CD55 and CD59 accounts for the intravascular hemolysis. Eculizumab is a humanized monoclonal antibody against complement component 5 (C5), thereby inhibiting terminal complement-mediated hemolysis. Eculizumab treatment has significantly reduced hemolysis and transfusion dependency, and improved anemia. However, the hematologic benefit varies considerably among patients. Lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low level hemolysis. Remarkable extravascular hemolysis mediated by C3 opsonization of PNH red blood cells (RBCs) in some patients with PNH receiving eculizumab has been reported (Blood 113,4094,2009). Complement receptor type 1 (CR1) has a crucial role in dissociation of C3/C5 convertases which activate C3. To explain the mechanism of extravascular hemolysis mediated by C3 opsonization in patients with PNH receiving eculizumab, the kinetics of CR1 and C3 in six patients with PNH (UPN2 to 7) who are treated with eculizumab at Osaka University Hospital were analyzed (Table1). We first confirmed that patients with PNH (UPN2 to 5) being treated with eculizumab had C3 positive RBCs with variable degree (1.8%-39.8%) and C3 positive RBCs were solely identified within CD59 negative RBCs, whereas healthy controls had only CD59 positive/C3 negative RBCs and untreated patients had both CD59 positive and negative/C3 negative but no C3 positive RBCs. Certain number of C3 positive RBCs were identified from immediately after the first initiation dose of eculizumab, and the proportion of C3 positive RBCs continuously increased with 14 weeks time course in two patients (UPN6 and 7) who could be monitored from before treatment. Then, the proportion of C3 positive RBCs was monitored at the interval of 15 months during treatment, and the proportion of C3 positive RBCs increased in all patients (Figure 1). At this moment, no one had significant clinical symptoms of extravascular hemolysis, and only UPN3 and 4 showed weak positivity in conventional direct anti-globulin test (DAT) at some points. However, DAT using monospecific anti-C3b plus anti-C3d antibody or anti-C3d antibody alone became positive in five out of six cases except for UPN2. Since CR1 has a crucial role in dissociation of C3/C5 convertases, especially under the conditions of CD55 deficiency, such as PNH, we analyzed the expression levels of CR1 on RBCs. The expression levels of CR1 on RBCs negatively well correlated with the percentage of C3 positive RBCs (Correlation=-0.979) (Figure 2). Since allelic variant of CR1 was known, we screened six patients and no homozygous variant known to express low levels of CR1 was identified. Only heterozygous variant was identified in UPN5 who express slightly low levels of CR1 compared to control (0.47). Then, the kinetics of CR1 and C3 during RBC maturation were analyzed in fractions of reticulocytes and mature RBCs. The proportion of C3 positive RBCs tended to increase, whereas the expression of CR1 tended to decrease during RBC maturation in four patients (UPN3,4,5 and 7). Thus, we confirmed that patients with PNH started to have C3 positive RBCs mediated by the treatment of eculizumab without exception, and the proportion of C3 positive RBCs increased during treatment. Most patients showed positive DAT using monospecific anti-C3b/anti-C3d antibody, even if there is no significant clinical symptoms of extravascular hemolysis. We also identified that the patients expressing high levels of CR1 tended to have low proportion of C3 positive RBCs, and the expression of CR1 tended to decrease with increasing the proportion of C3 positive RBCs during RBC maturation. We have successfully established several methods to monitor the kinetics of CR1 and C3, although current data are still not conclusive because analyzed patients had little symptoms. Therefore, we have conducted the research project to recruit the patients having significant clinical symptoms of extravascular hemolysis. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.

scholarly journals Glycosylation of gp41 of Simian Immunodeficiency Virus Shields Epitopes That Can Be Targets for Neutralizing Antibodies

2008 ◽  
Vol 82 (24) ◽  
pp. 12472-12486 ◽  
Author(s):  
Eloìsa Yuste ◽  
Jacqueline Bixby ◽  
Jeffrey Lifson ◽  
Shuji Sato ◽  
Welkin Johnson ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
Transmembrane Protein ◽  
Glycosylation Site ◽  
Parental Virus ◽  
Recognition Sequence ◽  
Glycosylation Sites ◽  
Immunodeficiency Virus ◽  
Protein Gp41

ABSTRACT Human immunodeficiency virus type 1 and simian immunodeficiency virus possess three closely spaced, highly conserved sites for N-linked carbohydrate attachment in the extracellular domain of the transmembrane protein gp41. We infected rhesus monkeys with a variant of cloned SIVmac239 lacking the second and third sites or with a variant strain lacking all three of SIVmac239's glycosylation sites in gp41. For each mutation, asparagine (N) in the canonical N-X-S/T recognition sequence for carbohydrate attachment was changed to the structurally similar glutamine such that two nucleotide changes would be required for a reversion of the mutated codon. By 16 weeks, experimentally infected monkeys made antibodies that neutralized the mutant viruses to high titers. Such antibodies were not observed in monkeys infected with the parental virus. Thus, new specificities were revealed as a result of the carbohydrate attachment mutations, and antibodies of these specificities had neutralizing activity. Unlike monkeys infected with the parental virus, monkeys infected with the mutant viruses made antibodies that reacted with peptides corresponding to the sequences in this region. Furthermore, there was strong selective pressure for the emergence of variant sequences in this region during the course of infection. By analyzing the neutralization profiles of sequence variants, we were able to define three mutations (Q625R, K631N, and Q634H) in the region of the glycosylation site mutations that conferred resistance to neutralization by plasma from the monkeys infected with mutant virus. Based on the reactivity of antibodies to peptides in this region and the colocalization of neutralization escape mutations, we conclude that N-linked carbohydrates in the ectodomain of the transmembrane protein shield underlying epitopes that would otherwise be the direct targets of neutralizing antibodies.

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.

scholarly journals Human lysosomal α-glucosidase: functional characterization of the glycosylation sites

1993 ◽  
Vol 289 (3) ◽  
pp. 681-686 ◽  
Author(s):  
M M P Hermans ◽  
H A Wisselaar ◽  
M A Kroos ◽  
B A Oostra ◽  
A J J Reuser
Keyword(s):  
Functional Characterization ◽  
Enzyme Synthesis ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Post Translational Modification ◽  
Transport Pathway ◽  
Glycosylation Sites ◽  
Mature Enzyme ◽  
Alpha Glucosidase

N-linked glycosylation is one of the important events in the post-translational modification of human lysosomal alpha-glucosidase. Phosphorylation of mannose residues ensures efficient transport of the enzyme to the lysosomes via the mannose 6-phosphate receptor. The primary structure of lysosomal alpha-glucosidase, as deduced from the cDNA sequence, indicates that there are seven potential glycosylation sites. We have eliminated these sites individually by site-directed mutagenesis and thereby demonstrated that all seven sites are glycosylated. The sites at Asn-882 and Asn-925 were found to be located in a C-terminal propeptide which is cleaved off during maturation. Evidence is presented that at least two of the oligosaccharide side chains of human lysosomal alpha-glucosidase are phosphorylated. Elimination of six of the seven sites does not disturb enzyme synthesis or function. However, removal of the second glycosylation site at Asn-233 interferes dramatically with the formation of mature enzyme. The mutant precursor is synthesized normally and assembles in the endoplasmic reticulum, but immunoelectron microscopy reveals a deficiency of alpha-glucosidase in the Golgi complex and in the more distal compartments of the lysosomal transport pathway.

scholarly journals Novel Mutations in the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Type 1 Causing Blomstrand Osteochondrodysplasia Types I and II

2007 ◽  
Vol 92 (3) ◽  
pp. 1088-1095 ◽  
Author(s):  
J. Hoogendam ◽  
H. Farih-Sips ◽  
L. C. Wÿnaendts ◽  
C. W. G. M. Löwik ◽  
J. M. Wit ◽  
...  
Keyword(s):  
Splice Site ◽  
Residual Activity ◽  
Severe Form ◽  
Dermal Fibroblasts ◽  
Receptor Type ◽  
Type I ◽  
Type Ii ◽  
Complete Inactivation ◽  
Low Levels

Abstract Context: The PTH/PTHrP receptor type 1 (PTHR1) has a key role in endochondral ossification, which is emphasized by diseases resulting from mutations in the PTHR1 gene. Among these diseases is Blomstrand osteochondrodysplasia (BOCD). Objective: BOCD can be divided into two types, depending on the severity of the skeletal abnormalities. The molecular basis for this heterogenic presentation is unknown. Design and Patients: We performed mutation analysis in two families with type I and in three families with the less severe form of BOCD type II. Results: In one of the type I BOCD cases, a homozygous nonsense mutation (R104X) was found, resulting in a truncated PTHR1. In the second type I BOCD case, no mutation was found. A homozygous nucleotide change (intron M4+27C>T) was demonstrated in one of the type II BOCD cases creating a novel splice site. In dermal fibroblasts of the patient, this novel splice site was preferentially used, resulting in an aberrant transcript. The wild-type transcript remained, however, present, albeit at low levels. In the other two families with type II BOCD, a previously identified homozygous missense mutation (P132L) was found. Functional analysis demonstrated that the P132L mutant had low residual activity. Conclusions: In combination with data presented in literature, we conclude that type I BOCD is caused by a complete inactivation of the PTHR1, whereas low levels of residual activity due to a near complete inactivation of the PTHR1 result in the relatively milder presentation of type II BOCD.

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.

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