A naturally occurring human Nedd4–2 variant displays impaired ENaC regulation in Xenopus laevis oocytes

2004 ◽  
Vol 287 (3) ◽  
pp. F550-F561 ◽  
Author(s):  
Fatemeh Fouladkou ◽  
Rasoul Alikhani-Koopaei ◽  
Bruno Vogt ◽  
Sandra Y. Flores ◽  
Laurence Malbert-Colas ◽  
...  
Keyword(s):  
Expression System ◽  
Donor Site ◽  
Susceptibility Gene ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Nonsynonymous Mutation ◽  
Single Strand Conformational Polymorphism ◽  
Phosphorylation Level ◽  
Xenopus Oocyte Expression

The epithelial Na+ channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4–2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/deleted in Liddle's syndrome, resulting in elevated Na+ reabsorption and hypertension explained partly by impaired ENaC-Nedd4–2 interaction. We hypothesized that Nedd4–2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4–2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4–2-P355L) was further characterized. This mutation next to the 3′ donor site of exon 15 does not affect in vitro splicing of Nedd4–2 mRNA. However, in the Xenopus oocyte expression system, Nedd4–2-P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4–2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4–2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4–2 Ser448 (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer448 antibody, we observed stronger basal phosphorylation of Nedd4–2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na+ currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4–2 provides an explanation for the different potential to inhibit ENaC activity.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

scholarly journals Gain-of-function variant of the human epithelial sodium channel

2013 ◽  
Vol 304 (2) ◽  
pp. F207-F213 ◽  
Author(s):  
Jingxin Chen ◽  
Thomas R. Kleyman ◽  
Shaohu Sheng
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Surface Expression ◽  
Wild Type ◽  
Open Probability ◽  
Gain Of Function ◽  
Human Disorders ◽  
Human Genome Sequencing ◽  
Xenopus Oocyte Expression ◽  
Salt Sensitive Hypertension

Epithelial Na+ channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis, and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing αβγL511Q human ENaCs showed 4.6-fold greater amiloride-sensitive currents than cells expressing wild-type channels. The γL511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had fourfold higher open probability than wild type. In addition, γL511Q largely eliminated the Na+ self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na+. Moreover, γL511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that γL511Q is a gain-of-function human ENaC variant. Our results suggest that γL511Q enhances ENaC activity by increasing channel open probability and dampens channel regulation by extracellular Na+ and proteases.

Biophysical effects of pore mutations of ROMK1

2001 ◽  
Vol 101 (2) ◽  
pp. 121-130 ◽  
Author(s):  
Sunil BHANDARI ◽  
Malcolm HUNTER
Keyword(s):  
Expression System ◽  
Structural Elements ◽  
Loss Of Function ◽  
Wild Type ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Functional Features ◽  
Living Organisms ◽  
Pore Lining ◽  
Channel Properties

Potassium channels are ubiquitous, being present in all living organisms. These proteins share common structural elements, which confer common functional features. In general, all K+ channels have a high selectivity for K+, and are blocked by cations of similar dimensions, such as Cs+ and Ba2+. Mutations in the pore region tend to lead to either the total loss of function or K+ selectivity. We have made mutations to one of the most highly conserved residues of the pore, glycine-143, of the inward rectifier ROMK1 (Kir1.1), and examined the resulting channel properties in the Xenopus oocyte expression system with a two-electrode voltage clamp. Mutations G143A and G143R resulted in failure to express functional channels. Co-injection of wild-type ROMK1 cRNA with these mutants led to rescue of channel function, which was different from wild-type ROMK1. In both mutants, the sensitivity to Ba2+ and Cs+ was increased, the rate of onset of block by Ba2+ was enhanced, and the selectivity to potassium was reduced. Whereas the crystallographic evidence shows that cations bind to the carbonyl backbone of the pore-lining residues, the present results indicate that the side chains of these amino acids, which face away from the pore lining, also affect permeation.

scholarly journals Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness

2007 ◽  
Vol 293 (1) ◽  
pp. C337-C345 ◽  
Author(s):  
Dwan A. Gerido ◽  
Adam M. DeRosa ◽  
Gabriele Richard ◽  
Thomas W. White
Keyword(s):  
Skin Disease ◽  
Expression System ◽  
Fatal Outcome ◽  
Gjb2 Gene ◽  
Wild Type ◽  
Human Phenotype ◽  
Extracellular Milieu ◽  
Xenopus Oocyte Expression ◽  
Functional Consequences ◽  
Intercellular Channels

Mutations in the human GJB2 gene, which encodes connexin26 (Cx26), underlie various forms of hereditary deafness and skin disease. While it has proven difficult to discern the exact pathological mechanisms that cause these disorders, studies have shown that the loss or abnormal function of Cx26 protein has a profound effect on tissue homeostasis. Here, we used the Xenopus oocyte expression system to examine the functional characteristics of a Cx26 mutation (G45E) that results in keratitis-ichthyosis-deafness syndrome (KIDS) with a fatal outcome. Our data showed that oocytes were able to express both wild-type Cx26 and its G45E variant, each of which formed hemichannels and gap junction channels. However, Cx26-G45E hemichannels displayed significantly greater whole cell currents than wild-type Cx26, leading to cell lysis and death. This severe phenotype could be rescued in the presence of elevated Ca2+levels in the extracellular milieu. Cx26-G45E could also form intercellular channels with a similar efficiency as wild-type Cx26, however, with increased voltage sensitive gating. We also compared Cx26-G45E with a previously described Cx26 mutant, A40V, which has an overlapping human phenotype. We found that both dominant Cx26 mutants elicited similar functional consequences and that cells coexpressing mutant and wild-type connexins predominantly displayed mutant-like behavior. These data suggest that mutant hemichannels may act on cellular homeostasis in a manner that can be detrimental to the tissues in which they are expressed.

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

2002 ◽  
Vol 363 (2) ◽  
pp. 353-358 ◽  
Author(s):  
Toni PETAN ◽  
Igor KRIŽAJ ◽  
Franc GUBENŠEK ◽  
Jože PUNGERČAR
Keyword(s):  
Expression System ◽  
Enzymic Activity ◽  
Terminal Region ◽  
Wild Type ◽  
Considerable Decrease ◽  
Phospholipases A2 ◽  
Neuronal Proteins ◽  
Interfacial Adsorption ◽  
Vipera Ammodytes

Ammodytoxins (Atxs) are group II phospholipases A2 (PLA2s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA2 toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe24, in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp24 mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40–80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA2 activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe24 also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn24 mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe24 in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

2020 ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Km Value ◽  
Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.

Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product

1990 ◽  
Vol 10 (7) ◽  
pp. 3761-3769
Author(s):  
W G Kaelin ◽  
M E Ewen ◽  
D M Livingston
Keyword(s):  
Gene Product ◽  
Structural Information ◽  
Susceptibility Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Adenovirus E1a

It has previously been demonstrated that the simian virus 40 large T antigen and adenovirus E1A proteins can form complexes with the retinoblastoma susceptibility gene product (RB). We studied the ability of these proteins to bind to mutant RB proteins in vitro. A region of RB spanning residues 379 to 792 was found to be both necessary and sufficient for binding to T or E1A. Furthermore, this region of RB contains sufficient structural information to mimic wild-type RB in its ability to distinguish between wild-type T and the transformation-defective T mutant K1. The results of competition experiments with peptide analogs of the RB-binding sequence in T suggest that this region of RB makes direct contact with a short colinear region of T, i.e., residues 102 to 115, previously implicated in both transformation and RB binding.

scholarly journals Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao-Yang Zhou ◽  
Xiang-Ran Lu ◽  
Ying-Hui Li ◽  
Ya-Qing Ma ◽  
Shi-Wen Zhao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Enzymatic Activity ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
High Dose ◽  
Nucleotide Position ◽  
The Novel ◽  
Wild Type

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

scholarly journals Genetic Analysis of the Dihydrofolate Reductase-Thymidylate Synthase Gene from Geographically Diverse Isolates of Plasmodium malariae

2007 ◽  
Vol 51 (10) ◽  
pp. 3523-3530 ◽  
Author(s):  
Naowarat Tanomsing ◽  
Mallika Imwong ◽  
Sasithon Pukrittayakamee ◽  
Kesinee Chotivanich ◽  
Sornchai Looareesuwan ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Thymidylate Synthase ◽  
Plasmodium Malariae ◽  
Drug Susceptibility ◽  
Wild Type ◽  
Nonsynonymous Mutation ◽  
Gene Coding ◽  
Antifolate Resistance ◽  
Plasmodium Brasilianum

ABSTRACT Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).

scholarly journals Glycosylation Tunes Neuroserpin Physiological and Pathological Properties

2020 ◽  
Vol 21 (9) ◽  
pp. 3235 ◽  
Author(s):  
Cristina Visentin ◽  
Luca Broggini ◽  
Benedetta Maria Sala ◽  
Rosaria Russo ◽  
Alberto Barbiroli ◽  
...  
Keyword(s):  
Pathological Condition ◽  
Expression System ◽  
Point Mutations ◽  
Tryptophan Fluorescence ◽  
Biochemical Properties ◽  
Eukaryotic Expression ◽  
Cell Models ◽  
Wild Type

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

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