Biophysical characterization of KO513, a protein expressed in human invasive glioblastoma

Abstract

Glioblastoma multiforme (GBM) is the deadliest brain tumour. GBM is associated with poor prognosis, with its patients having a very short median survival and poor response to chemotherapy. GBM is a World Health Organization (WHO) grade IV glioma and accounts for up to 78% of all brain tumours. This phenotype harbours a series of mutations that provide cells with selective growth advantages that promote survival and proliferation in a hostile and hypoxic environment. KIAA0513 is one of the genes identified to be upregulated in GBM phenotype through gene expression profiling. KIAA0513 gene codes for K0513 protein. KIAA0513 is ubiquitously expressed and is enriched in the cerebral area of the brain. It is predicted to be involved in signalling pathways including neuroplasticity, cytoskeletal regulation and apoptosis. The main objective of the current study was to perform biophysical characterization of K0513. Characterization of K0513 will allow structure-function annotation which may serve as a basis for establishment of K0513 as a potential biomarker for GBM. Using bioinformatics analysis, a potentially functional SBF2 domain was identified. The threedimensional homology model shows that K0513 is a globular protein, with the identified SBF2 domain and transmembrane region in proximity to one another. Predicted interacting partners includes members of the Rab GTPase family, membrane proteins, transcription factors and neurotransmitters. The overall in silico analysis suggest that K0513 may possess nucleotide exchange factor (NEF) activity for Rab3a. Recombinant proteins, non-codon harmonized (K0513W) and codon harmonized (K0513H) were expressed in E. coli XL1-Blue cells. The recombinant K0513H was successfully purified using nickel-affinity chromatography, this is the first study to report on the recombinant expression and purification of K0513. Using tryptophan fluorescence assay and limited proteolysis, nucleotides were found to have no significant effect on the tertiary structural conformation of K0513. Pull down assay shows promising interactors of K0513 from fibrosarcoma cell line; future studies will identify the interactors using Liquid chromatography-mass spectrometry.