Obtaining And Identification Of The Gene Encoding A Vitiligo Associated Melanocyte Autoantigen VIT 75

The pathogenesis of vitligo, an acquired hypomelantic disorder resulting from the loss of functional melanocytes, is not fully defined yet. Four major hypotheses for the pathogenesis of vitiligo have been proposed, including neural hypothesis, autoimmune aetiology, self-destruction of pigment cells, genetic theory. In resent years,autoimmunity were revealed as the most important causative factor to vitiligo. The frequent association of vitiligo with other autoimmune diseases, together with many studies demonstrating that vitiligo patients have autoantibodies against melanocyte antigens add support to the autoimmune theory. Several specific vitiligo autoantigens have been reported to be targets of autoantibodies, which are tyrosinase, tyrosinase related protein 1(TRP-1),tyrosinase related protein 2(TRP-2),and melanin- concentrating hormone receptor 1(MCHR-1). So far, none of these has been unequivocally recognized as the major autoantigens in the disease. Initial studies revealed that antibodies in vitiligo patients were most directly against pigment cell antigens with molecular weights of 35, 40-45, 75, 90 and 150 kDa. Several of the proteins (40-45, 75 and 150 kDa) appeared to be common tissue antigens, while others (35 and 90 kDa) were preferentially expressed on pigment cells. Up to date, melanocyte antigens of 75, 90 kDa were not identified. They were named as VIT 75, VIT 90 respectively.The previous study of our team showed that the pI of the melanocyte autoantigen with VIT 75 was 6.5. 83% of vitiligo patients were considered positive for anti-VIT 75 antibodies. Our own work also detected VIT 75 antibodies in vitiligo patients were prominently higher than other autoantibodies. So it was important to identify the cDNA of VIT 75, which will help us to better understand immunity mechanisms of vitiligo.Objective: Present study is to isolate and identify VIT 75 by 2-dimensional electrophoresis and western blot. The aim of this study is to identify the encoding gene of VIT 75 by mass spectrum and bioinformatics.Methods:[1] Identification of vitiligo associated melanocyte auto -antigen VIT 75 We cultured the epidermic melanocye cell and preparated the membrane fractions and proteins separation. The membraneproteins were segregated by 2-dimensional electrophoresis(2-DE), The western blot analysis resluts were compared between vitiligo patients and control. VIT 75 was discriminated. [2] Identification for the amino acid sequence of VIT 75 by mass spectrum and bioinformatics To identify the the amino acid sequence of VIT 75, we used MALDI-TOF/TOF method. The result of the mass spectrum analysis was used to search the NCBInr database using the Mascot search engine. We used soft ware on line to analysis the amino acid sequence of candidate protein, to evaluate the potential constitutions of the candidate protein. [3] Identifition of mitofilin by RT-RCR,western blot and immunocytochemistry According to the nucleotide sequence of the candidate protein, we design the primers. Detected the mRNA and protein level expression of candidate protein by RT-RCR,western blot and immunocytochemistry respectively.Results:[1] Identification of vitiligo associated melanocyte auto -antigen VIT 75 The membraneproteins of melanocyte were segregated by 2-dimensional electrophoresis(2-DE). The western blot analysis resluts that compared between vitiligo patients and control showed that there was a different spot in the gel. The PI of VIT 75 was 6.5. The resulte was consistented with our previous result.[2] Identification for the amino acid sequence of VIT 75 by mass spectrum and bioinformaticsThe result of the mass spectrum analysis was used to search the NCBInr database using the Mascot search engine. There were eight proteins containing four strips of sequenced amino acid sequence. Contrasted the amino acid sequence of candidate proetins by DNAMAN soft ware, we found that these proetins were highly similar in amino acid sequence. Searched the NCBInr database, we found that these proetins all were productions of the same CDs. The geneID is 10989. Given these above results, we preliminarily considered the inne membrane protein, mitofilin, as candidate protein for VIT 75. To evaluate the potential constitutions of mitofilin, we used soft ware online to analysis the amino acid sequence of mitofilin.The results suggested that mitofilin has transmembrane motif and the amino acid sequence of mitofilin contained many potential phosphorylation site, but not glycosylation site.[3] Identifition of mitofilin by RT-RCR,western blot and immunocyto - chemistryTo confirm the relationship between VIT 75 and mitofilin ,we performed western blot. The reslut of western blot showed that VIT 75 was mitofilin. The results of RT-RCR analysis showed that mitofilin were expressed in melanocyte,melanoma cell line 11147 and HaCaT cell lines at the mRNA level. The expression of mitofilin at protein level was dectected by western blot. The results showed that the expression of mitofilin was higher in melanocyte than in HaCaT. Immunocytochemistry showed that mitofilin expressed in cytoplasm of HaCaT, in contrast, it expressed both cytoplasm and membrane of melanocyte.Conclusions: 1. For the first time, we obtained the amino acid sequence of VIT 75, and identifed that VIT 75 was mitofilin. We predicted the modifitions of mitofilin by bioinformatics. We found that there were a transmembrane motif and many phosphorylation sites. 2. For the first time, we found antibodies against the inner membrane protein of mitochondria, mitofilin, in the serum of vitiligo patients. The present study represents a new proof and addition to the immunity theory of vitiligo.