Expression And Significance Of OTU1 In Mesangial Cell And Preparation Of Rabbit Anti-OTU1 Polyclonal Antibodies

Ubiquitin-proteasome pathway emerged as an important mechanism for regulating intracellular protein metabolism. It plays a key role in many cellular processes including cell cycle progression, signal transduction, DNA repair, receptor function, development, differentiation and inflammation. Protein ubiquitination is a reversible process regulated by many factors involving some specific deubiquitinating enzymes(DUBs). DUBs are involved in regulation of ub-dependent pathway by direct or indirect association with the proteasomes. DUBs are also implicated in numerous biologically important pathways and are given attention by majority of scholars.Otubains are a recently identified family of DUBs that belong to the ovarian tumor superfamily of proteins. All of them have OTU domain of 130 amino acids that is highly conserved from yeast to mammals. The proteins contain the conserved cysteine, histidine and aspartate residues that define the putative catalytic triad of cysteine proteases. Recent studies show that OTU1 have multifunctional characteristics in vivo. In addition to participating in the regulation of protein ubiquitination degradation, OTU1 plays an important role in cell differentiation and cell proliferation. OTU1 is located at chromosomal positions 11q13.1. There is widespread expression of OTU1 in many human tissues. It has been reported that OTU1 can express in kidney tissue by RT-PCR and Western Blot. But it is unclear of concrete cellular location and function in kidney. Up to now, there are no related reports about its relationship to kidney diseases.Decorin is a small proteoglycan that is composed of core protein and glycosaminoglycan chain. Current study found that DCN had a variety of functions. Previous experiment in our group showed that DCN may also inhibit TGF-βsignaling by binding to TGF-βso that it interferes with binding to TGF-βreceptor complex. It plays an important role in reduction of ECM deposition in the glomerular nephritis, inhibition of MC proliferation and development of glomerular sclerosis. It is an important factor in antagonizing nephritis injury. Recently the study about metabolism and regulation of DCN has been deepened. Experiments show that there are two forms about the metabolisms of DCN. DCN secreted into the extracellular matrix can be degraded through lysosomal after endocytosis by fibroblasts. However, intracellular DCN under external factors, can be degraded by Ubiquitin proteasome pathway through ER stress. IP was used in our previous experiment to prove that DCN is degraded by Ubiquitin-proteasome pathway in MC. Therefore, further understanding regulative mechanism about DCN may play an important role in research about antagonizing glomerulonephritis and glomerulosclerosis.In our experiment about mass spectra with DCN IP gel, we found that there is high expression of OTU1 in MC, which indicate OTU1 may be combined with DCN. By now, there is no related research report. So studying further if OTU1 can affect DCN degradation will be important to learn nephritis mechanism.In our article, we confirmed there was expression of OTU1 in MC and high expression of OTU1 under stimulation of inflammatory factors by technologies such as RT-PCR, protein analysis and so on. Results by IP show that OTU1 can combine with DCN in MC and OTU 1 can also affect ubiquitinated process of DCN, which indicate OTU1 is a regulator of DCN. All of the results provide a basis for further study about significance of DCN in regulating glomerulonephritis. Part I Construction of eukaryotic expression plasmid pIRES2-EGFP-OTU1-myc and the establishment of MC line of OTU1 over-expressionObjectives To construct eukaryotic expression plasmid OTU1 and select MC with high expression of OTU1 for further study of OTU1 functionMethods PCR was employed to amplify the OTU1 gene fragment.OTU1 gene was then subcloned into the pIRES2-EGFP vector, The vector was transformed into E.coli DH5a. We verified it using enzyme digestion and sequencing analysis. OTU1-myc was stably transfected into cultured rat MC and positive clones stably expressing OTU1 were selected by G418 and fluorescent protein. The positive clones were determined by RT-PCR and Western Blot.Results We amplified 850bp OTU1 gene fragmen by PCR, PCR product was inserted into the pIRES2-EGFP vector and then transformed into E.coli DH5a. It was confirmed by restriction endonuclease and DNA sequence analysis. We constituted successfully pIRES2-EGFP-OTU1-myc eukaryotic expression vector. After transfection and selection, the clones were selected with fluorescent protein. We detected the myc proteins and OTU1 mRNA. Compared with untransfected MC, there are obvious expression of myc protein and OTU1 mRNA. We obtained stable clones of over-expression OTU1 after the plasmid pIRES2-EGFP-OTU1-myc was transfected into cultured rat MC.Conclusions We constituted successfully pIRES2-EGFP-OTU1-myc eukaryotic expression vector, which was stably transfected into cultured rat MC. The positive clones of over-expressing OTU1 were selected. and identified by RT-PCR and Western Blot.Part II The expression of OTU1 in mesangial cell during glumerulonephritides and its relationship to DCNObejectives To explore the expression of OTU1 in mesangial cell and in glomerulonephritides. And to investigate its significance in regulation of DCN degradation via ubiquitin-proteasome pathwayMethods RT-PCR, Western Blot and Immunofluorescence analysis were used to detect the gene transcription and protein expression of OTU1 in the cultured rat and human mesangial cell. Realtime PCR and Western Blot were employed to examine the change of mRNA and protein levels after the rat MC was treated by IL-1 P and ATS. The distribution of OTU1 expression in mesangium during glomerulonephritides was investigated in human renal needle biopsy tissue by immunohistochemistry. We observed the change of protein expression of DCN after the transfection. IP was performed to verify the interaction between OTU1 and DCN and to analyze the ubiquitination of DCN in the rat MC of OTU1 over-expression.Results There is the mRNA and protein expression of OTU1 in the rat and human MC. The gene transcription and protein expression of OTU1 were increased in the rat MC after 3h,6h and 12h of the stimulation by IL-1βand ATS. The OTU1 over-expression was seen in mesangial cell proliferative glomerulonephritides such as IgA nephropathy, LN and APGN, which imply the expression of OTU1 of MC may relate to the pathgenesis of glomerulonephritis. Transfection of OTU1 in MC increased protein expression of DCN and decreased the accumulation of ubiquitinated DCN. We verified that OTU1 can interact with DCN in MC by IP method.Conclusions OTU1 can express in cultured MC. Over-expression of OTU1 may increase DCN protein expression and reduce the level of ubiquitianted DCN.OTU1 and DCN can bind each other. It is probably that OTU1 is related to the development mechanisms of glomerulonephritis.Part III Preparation of anti-OTU1 polyclonal antibodyObjectives To prepare anti-OTU1 polyclonal antibody and preliminarily explore the expression of OTU1 in kidney tissue. Methods PCR was employed to amplify the OTU1 gene fragment. OTU1 gene was then subcloned into the pProExHTa vector, The construct vector was transformed into E.coli Rosette. We verified it using enzyme digestion and sequencing analysis. The protein was induced by IPTG and purified by Ni2+-NTA. SDS-PAGE and Western Blot were performed to assay the OTU1 protein. New Zealand rabbits were immunized by injection of recombinant purified OTU1-His fusion protein. Agar double diffusion test and indirect ELISA were used to detect the titer of anti-serum. We use Western Blot to examine the specificity. Immunohistochemistry was performed to assay the expression of OTU1 protein in kidney tissue by the polyclonal antibody against OTU1.Results We amplified 850bp OTU1 gene fragment from the template of vector pIRES2-EGFP-OTU1-myc with PCR, PCR product was inserted into the pProExHTa plasmid and then transformed into E.coli Rosette, it was confirmed by restriction endonuclease and DNA sequence analysis. We constituted successfully pProExHTa-OTU1 prokaryotic expression vector. After induced by IPTG and purified by Ni-NTA affinity chromatography, the fusion protein 6×His-OTU1 was expressed in E.coli Rosette using SDS-PAGE and Western Blot methods with a molecular weight of 36kD. The purified fusion protein was then used to immunize the New Zealand rabbits, we obtained antiserum against OTU1 with titer of 1:16000. We then verified its specificity by Western Blot. It has been determined the expression of OTU1 in kidney tissue by our home-made anti-OTU1 polyclone antibody with Immunohistochemistry.Conclusions By the methods of gene engineering, We constituted successfully pProExHTa-OTU1 prokaryotic expression vector. By the induction and purification, we obtained successfully the highly purified protein, then we used it to immunize rabbits and obtained the anti-OTU1 polyclonal antibody with high titer and specificity. We found that OTU1 can be expressed in renal glomerulus mesenterium with Immunohistochemistry method using our home-made antiserum.