Analyse And Identification Of Melanocyte Surface Antigens Associated With Vitiligo

Background Vitiligo is a common depigmented skin disorder. The mechanism is still unknown utterly. At present, there are some theory about vitiligo, such as immune hypothesis, genetic theory, autocytotoxic hypothesis and neural pathogensis. But each of them cann't explain completely characters of vitiligo. Therefore some experts suggested that vitiligo was a multi-factors involved disorder. The majority of investigation sustained the autoimmune hypothesis.Recent research showed that the pigmented lesions presented destruction of melanocytes and cellar infiltrating. The most direct and convincing evidence that vitiligo is autoimmune disease is that autoantibodies are present in the circulation of most patient with vitiligo, the level of vitiligo antibodies correlates with the extent of depigmentation and activity of the disease. Our group has succeeded to identify them by immunochemical analysis and demonstrate the functional capacity to kill pigment cells in vitro and do so by a mechanism-complement-dependent cytotoxicty. At present, the pigment cell antigens defined by antibodies in patients with vitiligo mainly are cytoplasmic proteins, such as tyrosinase, tyrosinase related protein-1(TRP1) and tyrosinase related protein-2 (TRP2). Other surface antigens still are still unknown.Although the mechanism is not clear on the antibodies recognize and immune response, one of the important roles is that antibodies inspire effect after binding to cell membrane proteins, not binding to the cytoplasmic proteins. In the general opinion, whole antibody cann't go into the cytoplasm. The known melanocyte cytoplasmic antigens cann't explain the immune mechanism. As far as the known the membrane antigens are concerned, these antigens are not clear, although one was identified for MCHR-1 through screening the cDNA library. The modeling peptide cann't reflect the space conformational epitopes and post-translational modification, such as glycogen or phosphoric acid epitope. And positive ratio is not high on vitiligo serum recognizing MCHR-1. Therefore, the investigation started proteins-performing functional unit. In order to seek for cell membrane proteins recognized for more vitiligo serum.We had confirmed the present of the anti-melanocytes antibodies in the serum through live cell ELISA, western blotting and immunoprecipitation. However they cann't separate the single protein availably, the character of there antigens were not clear. With the development on the proteomics, 2-DE and mass spectrum are perfecting gradually. These provide the opportunity for the purification and identification of proteins. Therefore, we would plan to investigate the protein tokens by proteomics technology, then verify the relativity with vitiligo, ultimately define the protein, which will lay a foundation for deeping into etiology of vitiligo.MethodsSection One Culture and identify Human Epidermal Melanocytes1. Culture and purify HEMn: Firstly, we cultured epidermic cell by the DMEM/F12 medium with CT and bFGF. Secondly, the medium changed into K-SFM for 3 days, in order to suppress the fibroblasts. Finally, passage celles were growing in the K-SFM medium with TPA and CT. Necessarily, added G418 in the medium can suppress the fibroblasts.2. Identify HEMn: The cultured cells were stained by S-100 and Dopa, since for special tyrosinase activity and protein marker in the melanocytes. Therefore, we can identify the property and purity of melanocytes.3. Culture and identify HEMn~DP: Pre-experiment indicated that positive dots in melanocytes sourced from darkly-pigmented skin were more than from common skin. Therefore, we cultured the human epidermal melanocytes isolated from darkly-pigmented neonatal foreskin by the same way and compared the tyrosinase activity and the growth curve with HEMn.Section Two Separate and analysis melanocyte membraneantigens associated with vitiligo by 2-DEl.Screen the higher tite vitiligo serum: Live cell ELISA screenedhigher tite vitiligo serum and HEMn-DP acted as substrate. Holographicproteins and the primary serum were run on the western blotting, in orderto gain higher and special vitiligo serum.2. Prepare membrane proteins: We improved the previous dissociationof membrane proteins, and extracted cell organelle by classificationmethod, and introduced CHAPS, thiourea, TBP et al.3.Separate proteins by 2-DE and western blotting: Firstly, Bradfordquantified the protein sample. The proteins run in the IPG strip for firstdimensional separation, then in the SDS-PAGE for second separation. Gel wasstained by sliver and saved. Secondly, prepare two parallel 2-DE, increasesample quantity, coomassie brilliant blue G250 staining, then westernblotting. Finally, compare the different dots between vitiligo and healthyserum.Section Three Mass spectrum and identification for difference proteins1. Mass spectrum: Difference protein dots were cut in the 2-DE, digested in situ, discolored. MALDI-TOF detected peptide finger map. All peaks must been corrected in order to eliminate enzyme self-cut apexes and keratin apexes before searching for proteins in proteome database.2. Sequencing for the protein: sequencing is the important method and idea for identification. One way is protein N-end sequencing. Difference protein dot was cut from gel, discolored, transferred into PVDF film and sequenced. Other way is Q-TOF sequencing. Preparation for sample wasthe same as N-end sequencing.Section Four Verify the relativity between Lamin A/C and vitiligo1. Detect apoptosis: HEMn-DP were digested at the time of second passage cell or sixth individually. The sample con was 1X105 / ml, and were detected apoptosis.2. Express the Lamin A/C: Then PCR was used to amplify the Lamin A/C coding region. The PCR product was cloned into pMD18—T plasmid and sequenced, then subcloned into vector pGEX—4T—1. The Lamin A/Cprotein was expressed in E coli of DH5 a as fusion protein with GSTinduced by IPTG. The fusion protein was purified by glutathione resincolumn.3. Analysis the relativity with vitiligo: After expression thallus wasgathered, they were used for Western blotting, which was for detectingthe relativity for vitiligo. Indirect ELISA detected serum antibodypositive ratio, the purified protein acting as the substrate.ResultsSection One Culture and identify Human Epidermal Melanocytes l.Culture and purify HEMn: The morphologic primary cell was characterized for multi-polarity, refraction, and liking fibroblasts. They changed to double polarity, slim and parallel array. The keratinocytes disappeared at the time of second passage.2. Identify HEMn: The cultured cell possessed the tyrosinase activity because for black Dopa buffer. And all of the cells were positive for Dopa and S-100 stain. Therefore, the cultured cells were melanocytes, and the purity can reach to 100%.3. Culture and identify HEMn-DP: There were no difference on the shape between HEMn-DP and HEMn. However, HEMn-DP possessed the characters that were longer sticking, higher culture condition, the higher tyrosinase activity and lower growth.Section Two Separate and analysis melanocyte membrane antigens associated with vitiligo by 2-DEl.Screen the higher tite vitiligo serum: 300 vitiligo sera was screened, we got 120 cases with positive result by live cell ELISA. We detected 66 sera of 120 cases by immunoblotting. The positive ratio was 87.9%(58/66) , comparatively 16.7% (3/18) for health case, there was significant different between vitiligo patients and the healthy (t=31.00, PO.01 ) . Antibodies could reacted with several antigens with approximate MWs of 150KEK 90 KD, 75 KEK 60 KD> 40-45 KEK 30KD. 2.Separate proteins by 2-DE and western blotting: We can extract membrane proteins from 1 X 107 cells. And these proteins focused between 4 and 7 on the isoelectric point by 2-DE, immunoblotting indicated 13 positive dots, 3 of 13 were difference dots between 3 mixed vitiligo serum and 3 healthy serum. One of three difference dots must be MCHR — 1 because for the same the isoelectric point and apparent molecular weight as in database. Section three Mass spectrum and identification for difference proteins1. Mass spectrum: PMF indicated the Ml digestion, low signal to noise rate and narrow apex. Actual value (1791.59 or 2162.92) neared to theory value (1791.66 or 2163.06), which acted as inner standard. Therefore, the PMF was ideal. As far as the searching results of protein B were concerned, the coverage rate reach to 35 % ,which can conclude it was TRP1.However, the coverage rate of protein A was only 29%, which cann't conclude that it was lamin A/C.2. Sequencing for the protein: N-end sequencing of protein A show us: N-end were Blocked. Remove the blocking was difficult, and apply for Q-TOF sequencing. The peptide that MW was 1307.15 was used into Q-TOF sequencing, it show us a six amino acid peptide (TLEGEL). BLAST provided one hundred proteins accord with the sequence. Therewere no the known antigens among the results.Section Four Verify the relativity between Lamin A/C and vitiligo1. Detect apoptosis: Second and sixth passage cell also presented apoptosis in some extent, there were 10% apoptosis cell in all live cell. And the rate in apoptosis rate for sixth passage cell was higher than for Second passage cell.2. Express the Lamin A/C: We successfully cloned the human Lamin A/C cDNA sequence, gene sequencing indicated that it was the same as reported sequence. And constructed fusion expressing vector(pGEX—4T — 1/ Lamin A/C. GST/ Lamin A/C fusion protein was correctly expressed.3. Analysis the relativity for vitiligo: 42 vitiligo serum were detected by western blotting, 3 serum was positive, comparably there were no positive serum in 12 healthy serum. We successfully purified GST/ Lamin A/C fusion protein, 48 vitiligo sera were detected by indirect ELISA, 8 serum was positive.Conclusion1. We established a mended method for culturing melanocytes. The cultured cells were melanocytes after passage, purification and identification. And HEMn-DP were cultured successfully, they possessed the characters that were longer sticking, higher culture condition, the higher tyrosinase activity and lower growth. These lay a foundation for next investigations.2. The HEMn-DP membrane proteins were extracted successfully, separated by 2-DE. Immunoblotting indicated three difference dots. One of three difference dots must be MCHR— I because for the same the isoelectric point and apparent molecular weight as in database. However the other two dots needed to be identified.3. Two proteins were detected by mass spectrum and sequencing. Searching and BLAST for proteome database showed that one protein was TRPl. Another was an unknown antigen. "Champion peptide" in...