scholarly journals Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.

1988 ◽  
Vol 8 (8) ◽  
pp. 3357-3363 ◽  
Author(s):  
K F Kozarsky ◽  
S M Call ◽  
S K Dower ◽  
M Krieger
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Interleukin 2 ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Structure Stability ◽  
Wild Type ◽  
Intracellular Sorting

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors

1988 ◽  
Vol 8 (8) ◽  
pp. 3357-3363
Author(s):  
K F Kozarsky ◽  
S M Call ◽  
S K Dower ◽  
M Krieger
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Interleukin 2 ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Structure Stability ◽  
Wild Type ◽  
Intracellular Sorting

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

scholarly journals Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1913-1920 ◽  
Author(s):  
M Ugorski ◽  
DP Blackall ◽  
P Pahlsson ◽  
SH Shakin-Eshleman ◽  
J Moore ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Blood Group ◽  
Cho Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Wild Type

Abstract Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.

scholarly journals Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1913-1920 ◽  
Author(s):  
M Ugorski ◽  
DP Blackall ◽  
P Pahlsson ◽  
SH Shakin-Eshleman ◽  
J Moore ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Blood Group ◽  
Cho Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glycophorin A ◽  
Blood Group Antigens ◽  
Wild Type

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.

scholarly journals Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis

1999 ◽  
Vol 339 (1) ◽  
pp. 185-192 ◽  
Author(s):  
Reika WATANABE ◽  
Kazuhito OHISHI ◽  
Yusuke MAEDA ◽  
Nobuo NAKAMURA ◽  
Taroh KINOSHITA
Keyword(s):  
Cell Surface ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Amino Acid Identity ◽  
Eukaryotic Cell ◽  
Surface Proteins ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Expression Cloning

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins. The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step. Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes. As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals The Low-Density Lipoprotein Class A Module of the Relaxin Receptor (Leucine-Rich Repeat Containing G-Protein Coupled Receptor 7): Its Role in Signaling and Trafficking to the Cell Membrane

Endocrinology ◽  
2007 ◽  
Vol 148 (3) ◽  
pp. 1181-1194 ◽  
Author(s):  
András Kern ◽  
Alexander I. Agoulnik ◽  
Gillian D. Bryant-Greenwood
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lipoprotein Class ◽  
Class A ◽  
Relaxin Family ◽  
G Protein Coupled ◽  
Relaxin Receptor

The relaxin receptor (LGR7, relaxin family peptide receptor 1) is a member of the leucine-rich repeat containing G protein-coupled receptors subgroup C. This and the LGR8 (relaxin family peptide receptor 2) receptor are unique in having a low-density lipoprotein class A (LDL-A) module at their N termini. This study was designed to show the role of the LDL-A in LGR7 expression and function. Point mutants for the conserved cysteines (Cys47 and Cys53) and for calcium binding asparagine (Asp58), a mutant with deleted LDL-A domain and chimeric LGR7 receptor with LGR8 LDL-A all showed no cAMP response to human relaxins H1 or H2. We have shown that their cell surface delivery was uncompromised. The mutation of the putative N-linked glycosylation site (Asn36) decreased cAMP production and reduced cell surface expression to 37% of the wild-type LGR7. All point mutant, chimeric, and wild-type receptor proteins were expressed as the two forms. The immature or precursor form of the receptor was 80 kDa, whereas the mature receptor, delivered to the cell surface was 95 kDa. The glycosylation mutant was also expressed as two forms with appropriately smaller molecular masses. Deletion of the LDL-A module resulted in expression of the mature receptor only. These data suggest that the LDL-A module of LGR7 influences receptor maturation, cell surface expression, and relaxin-activated signal transduction.

scholarly journals ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

2018 ◽  
Vol 23 (8) ◽  
pp. 823-831
Author(s):  
Puay-Wah Phuan ◽  
Guido Veit ◽  
Joseph-Anthony Tan ◽  
Ariel Roldan ◽  
Walter E. Finkbeiner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Nucleotide Binding ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Δf508 Cftr ◽  
Nucleotide Binding Domain 1 ◽  
Cftr Modulators

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

scholarly journals Loss of the C Terminus of Melanocortin Receptor 2 (MC2R) Results in Impaired Cell Surface Expression and ACTH Insensitivity

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5971-5971
Author(s):  
Andrea Hirsch ◽  
Eirini Meimaridou ◽  
Monica Fernandez-Cancio ◽  
Amit V. Pandey ◽  
María Clemente ◽  
...  
Keyword(s):  
Cell Surface ◽  
Severe Hypoglycemia ◽  
Surface Expression ◽  
Melanocortin Receptor ◽  
Cell Surface Expression ◽  
Wild Type ◽  
C Terminus ◽  
Exact Function ◽  
Acth Insensitivity ◽  
Glucocorticoid Deficiency

Objective: Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting: A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results: The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions: Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.

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