Kobayashi T, Urabe K, Orlow S J, Higashi K, Imokawa G, Kwon B S, Potterf B, Hearing V J
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Nov 18;269(46):29198-205.
The silver mutation in mice causes progressive graying of hair due to the loss of functional follicular melanocytes. Recently the silver locus gene (called Pmel 17) has been cloned; its encoded product shares homology with a chick melanosomal matrix protein and a bovine retinal pigment epithelial protein. Although the sequence of the silver gene and the correlation of its expression with pigment production have been reported, its function in melanogenesis is still unknown. In an effort to characterize that function, we have synthesized the predicted carboxyl-terminal peptide of the mouse Pmel 17 protein and generated a rabbit polyclonal antibody (alpha PEP13) to it; that antibody recognized the silver protein specifically. The immunoaffinity-purified silver protein lacked all of the known melanogenic catalytic activities which other tyrosinase-related proteins (TRP) have, nor did it appear to modulate any of those TRP activities. Metabolic labeling experiments demonstrated that the silver protein disappears in vivo within a few hours, indicating that it is rapidly degraded, or quickly processed to lose its carboxyl terminus. Cross-reactivity experiments showed that a recently reported anti-melanosomal matrix protein antibody (alpha MX) also recognizes the silver protein, although at a different epitope from that of alpha PEP13. Using Western immunoblotting, we analyzed subcellular fractions isolated from B16 F10 melanoma cells and found that the silver protein was rich in the melanosome fraction but was absent from coated vesicles which deliver TRPs to melanosomes. These results suggest that the silver locus product is a melanosomal matrix protein which may contribute to melanogenesis as a structural protein, although the possibility remains that it also has a novel catalytic function in melanogenesis.
小鼠中的银色突变会导致功能性毛囊黑素细胞丧失，从而使毛发逐渐变白。最近，银色位点基因（称为Pmel 17）已被克隆；其编码产物与鸡黑素体基质蛋白和牛视网膜色素上皮蛋白具有同源性。尽管已经报道了银色基因的序列及其表达与色素生成的相关性，但其在黑素生成中的功能仍然未知。为了表征该功能，我们合成了小鼠Pmel 17蛋白的预测羧基末端肽，并针对其产生了兔多克隆抗体（α-PEP13）；该抗体特异性识别银色蛋白。免疫亲和纯化的银色蛋白缺乏其他酪氨酸酶相关蛋白（TRP）所具有的所有已知黑素生成催化活性，它似乎也不能调节任何这些TRP活性。代谢标记实验表明，银色蛋白在体内几小时内就会消失，这表明它会迅速降解，或者很快被加工而失去其羧基末端。交叉反应实验表明，最近报道的一种抗黑素体基质蛋白抗体（α-MX）也能识别银色蛋白，尽管其表位与α-PEP13不同。我们使用蛋白质免疫印迹法分析了从B16 F10黑色素瘤细胞中分离出的亚细胞组分，发现银色蛋白在黑素体组分中含量丰富，但在将TRP转运到黑素体的被膜小泡中不存在。这些结果表明，银色位点产物是一种黑素体基质蛋白，它可能作为一种结构蛋白对黑素生成有贡献，尽管它在黑素生成中也具有新的催化功能的可能性仍然存在。
