title: Expression and secretion of wild type and mutant GNE proteins in Dictyostelium discoideum.
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.
authors.forename: Sonam
authors.initials: S
authors.lastname: Grover
authors.forename: Saima
authors.initials: S
authors.lastname: Aslam
authors.forename: Vijeta
authors.initials: V
authors.lastname: Sharma
authors.affiliation: School of Biotechnology, Jawaharlal Nehru University, New Delhi - 110067, India. ranjanaa@mail.jnu.ac.in.
authors.forename: Ranjana
authors.initials: R
authors.lastname: Arya
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009386:Neoplastic Syndromes, Hereditary; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D012878:Skin Neoplasms; D013217:Starvation
chemical_list: D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase