scholarly journals Molecular pathogenesis of human CD59 deficiency

2018 ◽  
Vol 4 (6) ◽  
pp. e280 ◽  
Author(s):  
Netanel Karbian ◽  
Yael Eshed-Eisenbach ◽  
Adi Tabib ◽  
Hila Hoizman ◽  
B. Paul Morgan ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Cell Surface ◽  
Chronic Inflammatory Demyelinating Polyneuropathy ◽  
Membrane Attack Complex ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Congenital Deficiency ◽  
Recurrent Strokes ◽  
And Function

ObjectiveTo characterize all 4 mutations described for CD59 congenital deficiency.MethodsThe 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality.ResultsImmunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation.ConclusionsAll 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.

scholarly journals Cell Surface Expression and Function of the Macromolecular C1 Complex on the Surface of Human Monocytes

2012 ◽  
Vol 3 ◽  
Author(s):  
Kinga K. Hosszu ◽  
Alisa Valentino ◽  
Yan Ji ◽  
Mara Matkovic ◽  
Lina Pednekar ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Monocytes ◽  
And Function

scholarly journals Intracellular Na+ Controls Cell Surface Expression of Na,K-ATPase via a cAMP-independent PKA Pathway in Mammalian Kidney Collecting Duct Cells

2003 ◽  
Vol 14 (7) ◽  
pp. 2677-2688 ◽  
Author(s):  
Manlio Vinciguerra ◽  
Georges Deschênes ◽  
Udo Hasler ◽  
David Mordasini ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Cell Surface ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cortical Collecting Duct ◽  
Mammalian Kidney ◽  
Duct Cells ◽  
Collecting Duct Cells

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K+-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.

scholarly journals Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

2016 ◽  
Vol 292 (4) ◽  
pp. 1524-1534 ◽  
Author(s):  
Stine Jørgensen ◽  
Christian Theil Have ◽  
Christina Rye Underwood ◽  
Lars Dan Johansen ◽  
Petrine Wellendorph ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Control Cell ◽  
Surface Expression ◽  
Genetic Variations ◽  
Cell Surface Expression ◽  
G Protein Coupled Receptor ◽  
Group 6 ◽  
And Function ◽  
G Protein Coupled

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

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 Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants

2000 ◽  
Vol 105 (7) ◽  
pp. 887-895 ◽  
Author(s):  
Jean-Pierre Morello ◽  
Ali Salahpour ◽  
André Laperrière ◽  
Virginie Bernier ◽  
Marie-Françoise Arthus ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Vasopressin Receptor ◽  
Pharmacological Chaperones ◽  
V2 Vasopressin Receptor ◽  
And Function

Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells

1994 ◽  
Vol 267 (3) ◽  
pp. F347-F353 ◽  
Author(s):  
M. D. Okusa ◽  
K. R. Lynch ◽  
D. L. Rosin ◽  
L. Huang ◽  
Y. Y. Wei
Keyword(s):  
Cell Surface ◽  
De Novo ◽  
Mdck Cells ◽  
Specific Binding ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Surface Binding ◽  
Binding Studies ◽  
Apical Domain

The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.

scholarly journals Nedd4-2 isoforms ubiquitinate individual epithelial sodium channel subunits and reduce surface expression and function of the epithelial sodium channel

2008 ◽  
Vol 294 (5) ◽  
pp. F1157-F1165 ◽  
Author(s):  
Nandita S. Raikwar ◽  
Christie P. Thomas
Keyword(s):  
Cell Surface ◽  
Sodium Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Ww Domains ◽  
Ubiquitin Proteasome ◽  
And Function

We previously reported the existence of multiple isoforms of human Nedd4-2 ( Am J Physiol Renal Physiol 285: F916–F929, 2003). When overexpressed in M-1 collecting duct epithelia, full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH2-terminal C2 domain (Nedd4-2ΔC2), and Nedd4-2 lacking WW domains 2 and 3 (Nedd4-2ΔWW2,3) variably reduce benzamil-sensitive Na+ transport. We investigated the effect of each of the Nedd4-2 isoforms on cell surface expression and ubiquitination of ENaC subunits. We find that αENaC when transfected alone or with β and γENaC is expressed at the cell surface and this membrane expression is variably reduced by coexpression with each of the Nedd4-2 isoforms. Nedd4-2 reduces the half-life of ENaC subunits and enhances the ubiquitination of α, β, and γENaC subunits when expressed alone or together suggesting that each subunit is a target for Nedd4-2-mediated ubiquitination. As has been reported recently, we confirm that the surface-expressed pool of ENaC is multi-ubiquitinated. Inhibitors of the proteasome increase ubiquitination of ENaC subunits and stimulate Na+ transport in M-1 cells consistent with a role for the ubiquitin-proteasome pathway in regulating Na+ transport in the collecting duct.

Sign in / Sign up

Export Citation Format

Share Document