Ultrasensitive Colorimetric Detection And Biological Function Study Of Protein O-GlcNAcylation

O-linked-N-acetyglucosamine(O-GlcNAc) is a dynamic and reversible protein post-translational modification similar to phosphorylation.O-Glc NAc transferase(OGT)and O-GlcNAcase(OGA)are two enzymes involved in this specific glycosylation.OGT is responsible for adding the GlcNAc group to the serine and threonine residues of the protein,while OGA removes the GlcNAc from the protein.O-GlcNAc modification occured mainly on the nucleus and cytoplasmic protein is different from traditional glycosylation.In addition,its sugar chain structure has only one oxadiazole linked N-acetylglucosamine monosaccharide.O-GlcNAc modification and phosphorylation directly or indirectly interact in cells,involved in cell proteolysis,transcriptional regulation,signal transduction,cell cycle regulation and other important life activities.In recent years,studies have shown that O-GlcNAcylation aberrant is involved in a variety of diseases such as tumor,neurodegenerative diseases,cardiovascular disease,diabetes and other diseases.Studies of O-GlcNAc glycosylatio modifications are of great importance in life sciences and biomedicine.Although researchers developed a variety of analytical methods,O-GlcNAc analysis has been greatly limited due to the low abundance and dynamic changes.The existing reports have mainly focused on the study of total intracellular O-GlcNAcylation level,which does not reflect the specific protein O-GlcNAc modification in tumorigenesis.In addition,the role in regulating the proliferation,migration and other related signal pathway of tumor cells and its molecular mechanism of O-GlcNAcylation also needs to be further studied.To overcome the challenges in the study of O-GlcNAcylation modification,wedeveloped a highly sensitive colorimetric dection method based on AuNP-catalyzed copper deposition-enabled efficient signal amplification.Furthermore,microarray chip technique was used to study the O-GlcNAc modification of specific proteins in prostate cancer cells.The effects of OGA inhibitors on proliferation and migration of prostate cells were also investigated.The main contents and results of this thesis are as follows:1.Construction and performance evaluation of a novel colorimetric method for O-Glc NAcylation detection based on copper deposition-enabled efficient signal amplificationIn this design,wheat germ agglutinin(WGA)was used as a specific recognition molecule for O-GlcNAc,and then colloidal gold nanoparticles(AuNP)were introduced onto the sensor surface through a biotin-streptavidin system.In the presence of the reducing agent ascorbic acid(AA)and copper sulfate(CuSO4),AuNP promotes the deposition of copper(Cu)on its surface.The amount of Cu deposited is proportional to the concentration of the analyte.The copper deposition catalyzed by AuNP(instead of expensive and unstable biological enzymes)can effectively amplify the detection signal to achieve an ultra-high detection sensitivity.After addition of ferric chloride(Fe Cl3),the deposited copper is oxidized to Cu2+ by ferric ions(Fe3+),while is reduced to ferrous ions(Fe2+)at the same time.Subsequently,the red complex is produced through the coordination between Fe2+and Bathophenanthrolinedisulfonic acid disodium salt,resulting in a change in the color of the solution.Thus,the level of protein O-GlcNAc modification can be qualitatively evaluated directly with the naked eye or be quantitatively detected by measuring the absorbance of the red solution.In this paper,N-acetyglucosamine conjugated-BSA(GlcNAc-conjugated BSA)was used as a model protein to evaluate the developed colorimetric assay.The results showed that the detection range of GlcNAc-conjugated BSA was 0.1 pg mL-1to 10 ng mL-1and limit of detection(LOD)was determined to be 0.02 pg mL-1.2.Application of the colorimetric detection method to evaluate the relationship between O-Glc NAc modification and tumorIn order to further evaluate the possibility of practical application of the above colorimetric detection,the O-GlcNAc levels in cell lysates of the prostate cancer cell line(PC-3)and the normal prostate cell line(RWPE-1)were studied.The results show that compared with the normal RWPE-1 cells,total O-GlcNAcylation level of cancer cell line PC-3 significantly increased,which is consistent with the reported results.3.The application of protein microarray chip in the evaluation of therelationship between O-GlcNAcylation and tumorThe results of protein microarray chip show that the level of O-GlcNAcylation in PC-3 is significantly higher than that in normal cell line RWPE-1.O-GlcNAcylation levels in PC-3,RWPE-1 cell lysate treated with OGA inhibitor Thiamet G,are higher than that of untreated cells,which confirms that the inhibition of OGA activity promotes the intracellular O-GlcNAc modification.In addition,compared with normal prostate cell RWPE-1,the expression level of OGT in prostate cancer cell PC-3 is significantly increased,while the expression level of OGA is decreased.These results also suggest that O-GlcNAc may be a good tumor biomarker and may play an important role in diagnosis and therapy of prostate cancer4.Antibody microarray and its application in parallel analysis of expression and O-GlcNAc modification of tumor-associated proteinsIn this work,c-Myc,NF-?B and p53 protein expression and O-GlcNAc modification in cell lysates were studied by using the antibody microarray chip.Compared with untreated cells,the levels of O-GlcNAc modification of c-Myc,NF-?B increased in cells treated with OGA inhibitor Thiamet G,whereas O-GlcNAc modification of p53 decreased,suggesting that OGA inhibitor could promote O-GlcNAcylation in c-Myc and NF-?B proteins and inhibit the O-GlcNAcylation of p53 protein.In comparison with normal prostate cells,the O-GlcNAc level of c-Myc and p53 decreased in prostate cancer PC-3 cells,while the O-GlcNAc level of NF-?B increased.5.Effect of OGA inhibitor on proliferation and migration of prostate cancer cellsIn order to further study the effect of O-GlcNAcylation on cell activities,cell proliferation and migration were evaluated by cell counting,MTT assay and cell scratch test.The results show that the level of O-GlcNAcylation is increased in cells treated with OGA inhibitor,and the cell proliferation is enhanced but the migration does not change significantly.In summary,this thesis project developed an ultrasensitive colorimetric detection technique based on AuNP-catalyzed copper deposition-enabled efficient signal amplification for low-abundance protein O-GlcNAc.We also studied O-GlcNAcylation of specific proteins in prostate cancer cells,discussed effect of OGA inhibitor on proliferation and migration of prostate cells.These results enrich the basic research of tumor and protein O-GlcNAcylation,and provide a theoretical basis for targeted therapyand drug development.