Journal ArticleDOI
Expression and Secretion of Wild Type and Mutant GNE Proteins in Dictyostelium discoideum
Reads0
Chats0
TLDR
Dd can be used as an expression host for the production of recombinant and functionally active form of GNE and its mutant proteins that can be use for biophysical characterization and structural determination of G NE to understand the pathomechanism of HIBM.Abstract:
GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) is a bifunctional enzyme which catalyzes the conversion of UDP-GlcNAc to ManNAc and ManNAc to ManNAc 6-phosphate, key steps in the sialic acid biosynthesis. Mutations in GNE lead to a neuromuscular disorder, Hereditary Inclusion Body Myopathy (HIBM). A major limitation in understanding the function of GNE is lack of recombinant full length GNE (rGNE) protein for detailed biophysical and structural characterization. In the present study, we have used Dictyostelium discoideum (Dd) as an alternate host for successful expression and secretion of functionally active form of GNE and its mutant proteins. We have generated Dd-AX3 stable cell lines harboring wtGNE or its mutants with Dd specific secretory signal sequence, PsA (prespore antigen). Upon starvation, rGNE was secreted in the medium from secretory vesicles. The rGNE was functionally active with epimerase activity (54±5.2 mU/mg) and kinase activity (66.45±3.48 mU/mg), while both epimerase and kinase activities of mutant GNE were drastically reduced. These activities were found to be statistically significant at p value < 0.05. Our study clearly demonstrates that Dd can be used as an expression host for the production of recombinant and functionally active form of GNE and its mutant proteins that can be used for biophysical characterization and structural determination of GNE to understand the pathomechanism of HIBM.read more
Citations
More filters
Journal ArticleDOI
GNE myopathy: from clinics and genetics to pathology and research strategies
Oksana Pogoryelova,José Andrés González Coraspe,Nikoletta Nikolenko,Hanns Lochmüller,Hanns Lochmüller,Andreas Roos +5 more
TL;DR: This review is summarising current GNE myopathy, scientific trends and open questions, which would be of significant interest for a wide neuromuscular diseases community.
Journal ArticleDOI
Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.
Sheng-Chia Chen,Chi-Hung Huang,Shu-Jung Lai,Chia Shin Yang,Tzu-Hung Hsiao,Ching-Heng Lin,Pin-Kuei Fu,Tzu-Ping Ko,Yeh Chen +8 more
TL;DR: Compared with the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria and full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme.
Journal ArticleDOI
Genetic defects in the hexosamine and sialic acid biosynthesis pathway
TL;DR: The biochemical mechanisms of known genetic defects in the hexosamine and CMP-sialic acid synthesis pathway in relation to the clinical phenotypes of congenital myasthenia, immunodeficiency or adult-onset myopathy are discussed.
Journal ArticleDOI
Glimpses of Dictyostelid research in India.
Rasheedunnisa Begum,Shweta Saran +1 more
TL;DR: Dictyostelium discoideum is a soil amoeba that undertakes a remarkable, facultative shift to multicellularity when exposed to starvation and requires signal pathways that result in alteration of gene expression and finally show cell differentiation.
Journal ArticleDOI
Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder.
TL;DR: In this paper , the effect of GNE mutation on its enzymatic activity and identification of potential small effector molecules was investigated. But, the study was limited to E. coli.
References
More filters
Journal ArticleDOI
A modified colorimetric method for the estimation of n-acetylamino sugars
Journal ArticleDOI
Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture
D. J. Watts,J. M. Ashworth +1 more
TL;DR: A simple axenic medium suitable for the growth of the myxamoebae of a strain of the cellular slime mould Dictyostelium discoideum is described and conditions suitable for initiating the cell differentiation of myxamonebae grown axenically are described.
Journal ArticleDOI
Reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of N-acetylglucosamine
TL;DR: Transport of the VSV-encoded glycoprotein between successive compartments of the Golgi has been reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc).
Journal ArticleDOI
The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy
Iris Eisenberg,Nili Avidan,Tamara Potikha,Hagit Hochner,Miriam Chen,Tsviya Olender,Mark Barash,Moshe Shemesh,Menachem Sadeh,Gil Grabov-Nardini,Inna Shmilevich,Adam Friedmann,George Karpati,Walter G. Bradley,Lisa Baumbach,Doron Lancet,Edna Ben Asher,Jacques S. Beckmann,Zohar Argov,Stella Mitrani-Rosenbaum +19 more
TL;DR: Findings indicate that GNE is the gene responsible for recessive HIBM, a unique group of neuromuscular disorders characterized by adult onset, slowly progressive distal and proximal weakness and a typical muscle pathology including rimmed vacuoles and filamentous inclusions.
Journal ArticleDOI
“Rimmed vacuole myopathy” sparing the quadriceps: A unique disorder in iranian jews
Zohar Argov,Rena Yarom +1 more
TL;DR: Nine cases from 4 different Iranian-Jewish families presented with generalized muscular weakness and Quadriceps muscle was the only leg muscle which retained its normal power and function even in advanced cases, suggesting an underlining neurogenic disorder.