Valencia Julio C, Rouzaud Francois, Julien Sylvain, Chen Kevin G, Passeron Thierry, Yamaguchi Yuji, Abu-Asab Mones, Tsokos Maria, Costin Gertrude E, Yamaguchi Hiroshi, Jenkins Lisa M Miller, Nagashima Kunio, Appella Ettore, Hearing Vincent J
Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2007 Apr 13;282(15):11266-80. doi: 10.1074/jbc.M608449200. Epub 2007 Feb 15.
Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed alphaPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. alphaPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with alphaPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using alphaPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.
Pmel17是一种黑素细胞/黑色素瘤特异性蛋白，对于黑素小体成熟形成成熟、纤维状且有色素的细胞器至关重要。最近，我们报道了糖基化程度较低的Pmel17形式（称为iPmel17）通过质膜的分选方式与成熟的Pmel17（称为mPmel17）不同，mPmel17直接分选至黑素小体。为阐明Pmel17独特加工和分选的机制，我们针对Pmel17重复结构域内对O-糖基化变化敏感的表位产生了一种高度特异性抗体（称为alphaPEP25h）。与识别iPmel17和mPmel17的抗体alphaPEP13h相比，alphaPEP25h仅识别iPmel17，并允许对iPmel17的加工和分选进行分析。我们使用alphaPEP25h的新发现表明，iPmel17不仅在内切糖苷酶H敏感性方面与mPmel17不同，而且在唾液酸修饰的核心1 O-聚糖含量上也不同。这一证据表明iPmel17在高尔基体中的糖基化方式不同，并且它通过分泌途径进行分选。对糖基化缺陷突变细胞中Pmel17加工的分析表明，缺乏正确添加唾液酸和半乳糖的Pmel17失去了形成纤维的能力。此外，我们表明添加唾液酸会影响Pmel17的稳定性和分选，并减少色素沉着。正常和转化的黑素细胞中唾液酸转移酶活性和底物的改变有所不同，这可能代表了恶性转化过程中的关键变化。
