Research On The Enrichment,identification Of O-GlcNAc Modification And Establishment Of Quantification Method

O-GlcNAcylation is an O-linked?-N-acetyl-glucosamine(O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays an important role in many significant cellular processes.Owing to its low molecular weight,uncharged property,and difficulty in distinguishing from?-N-acetyl-galactosamine(Gal NAc),the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions.Based on the identification and enrichment of O-GlcNAc modified proteins of CpOGA^D298N,comprehensive glycan binding profile of CpOGA^D298N have been identified.O-GlcNAc modified proteins have been identified from breast cancer cell line MCF-7 and Balb/c mouse liver.Considering the high specificity and avidity of CpOGA^D298Nand O-GlcNAc modification,proximity ligation assay(PLA)has been combined with CpOGA^D298N so as to estabilish a certain O-GlcNAc protein quantification method.Part?:Identification of glycan binding profile of CpOGA^D98N:We cloned and purified Clostridium perfringens OGA mutant(CpOGA^D298N),and sent the protein to the Consortium for Functional Gly Comics(CFG)for glycan miroarray binding identification.R software was used to draw the heat maps of glycan miroarray results of CpOGA^D298N,O-GlcNAc antibody CTD110.6 and Wheat germ agglutinin(Wheat germ agglutinin,WGA).The results comparison showed that the glycans combinded with CpOGA^D298N are mostly GlcNAc terminated,and Gal NAc terminated glycans show low affinity to CpOGA^D298N.Isothermal Calorimetry assay(ITC)also demonstrated the ability of CpOGA^D298N to distinguish between GlcNAc and Gal NAc.The above results primarily verified the high specificity and high affinity of CpOGA^D298Nbinding to O-GlcNAC modified proteins.Part II:Enrichment of O-GlcNAc modified proteins by CpOGA^D298N:Basis on the preliminary verification of high specificity and high affinity of CpOGA^D298Nand O-GlcNAc modification protein,we further using GST labeled CpOGA^D298N in western blot to identify O-GlcNAc proteins.Proper dosage of CpOGA^D298N used in far-western bolt was decided.And the monosaccharide GlcNAc can block the affinity of CpOGA^D298N and O-GlcNAc proteins.The staining of biotin labeled CpOGA^D298N on various cell surfaces was detected by flow cytometry,and it was found that CpOGA^D298N had merely no binding ability to cell surface proteins,while CpOGA^D298N had a high binding ability to the fixed and permeabilized cells,and the binding ability increased with the increase of the concentration of CpOGA^D298N.The results of laser confocal microscopy showed that all the CpOGA^D298N bound proteins were distributed inside of cells.The majority of O-GlcNAc modifications were distributed in the nucleus and cytoplasm,so the binding ability of CpOGA^D298N to cells was consistent with the distribution of O-GlcNAc modifications.84 high-confidence O-GlcNAc modified peptides of 82 proteins were enriched on the CpOGA^D298N-Sepharose 4b affinity column from breast cancer cell line MCF-7 and 33 high-confidence O-GlcNAc modified peptides of 33 proteins from Balb/c mouse liver tissue.Most of these proteins are newly identified O-GlcNAc modified proteins and do not bind Wheat Germ Agglutinin(WGA).Part III:Establishment of a new method for quantitative identification of O-GlcNAc modified proteins:In addition to the enrichment of O-GlcNAc modified peptide,a quantitative method for determining O-GlcNAc modified protein was established.Based on the binding ability of CpOGA^D298N to O-GlcNAc modified protein and the proximity ligation assay(PLA),this method was successfully applied to the quantitative detection of His tag labeled histone H2B in MCF-7 cells with a detection sensitivity as low as femto mole level.Empirically,this method can be used for the quantitative determination of any trace amount of O-GlcNAc modified proteins in the liquid by using a pair of appropriate protein-oligonucleotide conjugates(PLA probes).This quantification method provides a convenient and non-mass spectrometer-dependent method for the quantitative study of dynamic O-GlcNAc modification.