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J. Biol. Chem., Vol. 282, Issue 15, 11266-11280, April 13, 2007

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Sialylated Core 1 O-Glycans Influence the Sorting of Pmel17/gp100 and Determine Its Capacity to Form Fibrils^*

Julio C. Valencia¹, Francois Rouzaud, Sylvain Julien, Kevin G. Chen, Thierry Passeron, Yuji Yamaguchi, Mones Abu-Asab^¶, Maria Tsokos^¶, Gertrude E. Costin, Hiroshi Yamaguchi, Lisa M. Miller Jenkins, Kunio Nagashima^||, Ettore Appella, and Vincent J. Hearing²

From the Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, the Breast Cancer Biology Group, Cancer Research UK, Guy's Hospital, London SE1 9RT, United Kingdom, ^||Image Analysis Laboratory, NCI, National Institutes of Health, Frederick, Maryland 21702-1201, and the ^¶Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892

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 PEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. PEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with PEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using PEP25h 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.

Received for publication, September 1, 2006 , and in revised form, January 17, 2007.

^* This work was supported in part by the Intramural Research Program of the Center for Cancer Research, NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-8.

¹ To whom correspondence may be addressed: National Institutes of Health, Laboratory of Cell Biology, Bldg. 37, Rm. 2132, Bethesda, MD 20892-4256. Tel.: 301-496-1564; Fax: 301-402-8787; E-mail: valencij{at}mail.nih.gov.

² To whom correspondence may be addressed: National Institutes of Health, Laboratory of Cell Biology, Bldg. 37, Rm. 2132, Bethesda, MD 20892-4256. Tel.: 301-496-1564; Fax: 301-402-8787; E-mail: hearingv{at}nih.gov.

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