Characterization of three point mutations at the GlcNAc binding pocket of murine GlcNAc kinase and their effects on dendritic arborization

다국어 초록 (Multilingual Abstract)

N-acetylglucosamine (GlcNAc) kinase (NAGK; EC 2.7.1.59) converts GlcNAc into GlcNAc-6-phosphate. Based on the proposed 3D structure, we produced 3 point mutant NAGKs that were expected to retain differential capacities for substrate binding and reaction velocity. The proteins were expressed in Escherichia coli, affinity-purified to homogeneity, and used for functional analysis. Among the mutants, conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc to Ser (i.e., C143S) had the least affecton enzymatic activity. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc to Ala (i.e., N36A) mildly reduced the enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and acts as a proton acceptor to Ala (i.e., D107A), caused a total loss in the enzyme activity. The eGFP- or RFP (DsRed)-tagged mutant NAGKs increased the complexity of dendritic architecture when overexpressed in rat hippocampal neurons (DIV 5-9) with no statistical difference with wild-type NAGK. These results indicate that the upregulation of dendritic complex by NAGK is the enzyme’s non-canonical function.