The Identification Of O-GlcNAcylation Sites On HK2 And Generation Of HK2 Gene Kenockout Cell Lines

Glucose, protein and fat metabolism are the main modes of mammalian cells to obtain energy. The normal tissue is mainly relying on glucose metabolism through the glycolytic pathway to provide energy. Intermediate metabolites of glycolytic pathway which can provide raw materials for protein metabolism and fat metabolic pathway make the three major metabolic pathways closely linked. Glycolytic pathway has total of ten steps, containing three key kinases such as phospho fructokinase, hexokinase and pyruvate kinase, respectively. In the case of abundant oxygen, the three stages of aerobic respiration are aerobic glycolysis, the Krebs cycle and the electron transport system. During each stage, a number of chemical reactions take place which form the cellular respiration overall process. The outcome of aerobic glycolysis is that the glucose molecule is broken down into two pyruvate, or pyruvic acid molecules, which are broken down further in the Krebs cycle. Under the condition of hypoxia, pyruvate is eventually oxidated to lactic acid by glycolytic pathway, which is the anaerobic glycolysis pathway of the normal tissue. In the 1920s, Biologists Otto Warburg found that tumor cells under aerobic conditions could use glucose for glycolysis and produce lactic acid, namely Warburg effect.Hexokinase (HK) is one of key enzymes in the glycolytic pathway. In mammals, there exist four subtypes (HK1, HK2, HK3, HK4). In tumor cells, hexokinase type 2 (HK2) is related tightly to tumor and only HK2 is almost overexpressed. Our preliminary studies indicated that HK2 is modified by o-GlcNAc. O-GlcNAc modified proteins are involved in transcription, translation, cytoskeletal assembly, signal transduction, and many other cellular functions. Emerging research indicates that OGlcNAcylation is involved in many diseases, such as diabetes, cancer, alzheimer’s disease, heart disease and other neurodegeneration diseases.In the study, firstly we used E.coli as the prokaryotic expression system to express HK2 and OGT proteins. Then they were purified by affinity chromatography. The O-GlcNAc sites on HK2 were identified by MS spectrum when the protein was labeled by purified protein OGT. Finally, five O-GlcNAc sites were detected on HK2. These data would process the study of the function of O-GlcNAcylation. The glycosylation sites identification has a certain difficulty because 0-GlcNAc is modified by monosaccharide and it is easy hydrolysed. There may be false positive results in the five potential glycosylation sites; therefore we need to build eukaryotic expression vectors of HK2 which express muted HK2 on the O-GlcNAc sites. Eukaryotic expression vectors including mutated HK2 gene were transfected by lipofectamine 2000 into human embryonic kidney cells 293T, in order to further investigate the real OGlcNAcylation sites on HK2 in the cell.We need to get the stability cell lines of the endogenous HK2 knockout to study the HK2 glycosylation sites function. In recent years, with the development of biology related technology, CRISPR/Cas9 technical modification of the genome could provide a new train of thought. CRISPR/Cas9 technology is the third generation of artificial endonuclease technology which is more simple and ensitive compared to the Zinc finger endonuclease and activation of transcription factor effector nuc lease technology. This research adopted the CRISPR/Cas9 technology to knockout endogenous HK2 gene. First, we designed three fragment groups and then inserted them into the Cas9 carrier skeleton by enzyme digestion method and confirmed its sequence. The recombinant vectors were transfected by lipofectamine 2000 into breast cancer cells MDA-MB-231. We extracted the whole genomic DNA of breast cancer cells, amplified the target fragment by PCR and evaluated the efficiency of HK2 gene knockout through T7E1 enzyme digestion experiment. Then stable cell lines of HK2 knockout were established by limited dilution assay. Theses current results could provide useful foundation for further studies exploring the biological functions of HK2 O-GlcNAcylation in tumor.