Schiaffino M V, Baschirotto C, Pellegrini G, Montalti S, Tacchetti C, De Luca M, Ballabio A
Telethon Institute of Genetics and Medicine, San Raffaele Biomedical Science Park, Milan, Italy.
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9055-60. doi: 10.1073/pnas.93.17.9055.
Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.
1型眼白化病(OA1)是一种遗传性疾病,其特征为视力严重下降、畏光和视网膜色素减退。对皮肤黑素细胞和视网膜色素上皮进行超微结构检查发现存在巨大黑素小体,提示黑素小体生物发生存在缺陷。导致OA1的基因仅在色素细胞中表达,编码一种预测的404个氨基酸的蛋白质,该蛋白质具有几个假定的跨膜结构域,与先前鉴定的分子无相似性。我们使用多克隆抗体在视网膜色素上皮细胞、正常人黑素细胞和各种黑色素瘤细胞系中鉴定出了内源性OA1蛋白。通过蛋白质免疫印迹分析鉴定出两种形式的OA1蛋白,一种60 kDa的糖蛋白和一组48 kDa和45 kDa的蛋白条带,可能对应于未糖基化的前体多肽。在进行亚细胞分级分离并用非离子去污剂Triton X-114进行相分离后,OA1蛋白分离到富含黑素小体的部分,表现为一种真正的整合膜蛋白。对正常人黑素细胞进行免疫荧光和免疫金分析证实了内源性OA1蛋白在黑素小体膜上的定位,这与其可能参与黑素小体生物发生一致。鉴定出一种与人类疾病相关的新型黑素小体膜蛋白,将为控制亚细胞形态发生的细胞特异性途径的机制提供深入了解。