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.
Keywords: Dictyostelium discoideum, sialic acid, muscle myopathy, secretion, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine
kinase, hereditary inclusion body myopathy.
Rights & PermissionsPrintExport