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

Julio C. Valencia, 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 (+2 others)
2007 Journal of Biological Chemistry  
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
more » ... ific 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. The human PMEL17 gene encodes two type I integral membrane proteins that are translated from alternatively spliced mRNAs. Those two proteins, known as Pmel17 and gp100, differ as to whether they include a 7-amino acid motif in the mem-brane proximal region of the luminal domain (1-3). Pmel17 is critical to the formation of an internal fibrillar matrix within stage II melanosomes that is essential to the maturation of that organelle and the subsequent synthesis and deposition of melanin within it. Pmel17 is synthesized as a 68.6-kDa protein, as deduced from the cloned cDNA (2). It is rapidly and quantitatively glycosylated to an "immature" ϳ95-kDa form (termed iPmel17), but only a limited amount is further processed to a "mature" ϳ115-kDa form (termed mPmel17), due at least in part to N-glycosylation at five possible sites (4). mPmel17 can then be cleaved by a furin-like proprotein convertase that generates two fragments, one small fragment (cPmel17, ϳ26 kDa) containing the transmembrane (TM) 3 and the C-terminal domains, and the other larger fragment contains the N-terminal region (5). Those fragments can remain bound to each other by S-S bonds (5, 6). However, the iPmel17 formed remains fully endoglycosidase H (EndoH)-sensitive (4) and is significantly more stable over time than either of the other forms of Pmel17 (mPmel17 and cPmel17) (5). N-Linked core glycosylation begins in the ER and is essential for the function, stability, folding, intracellular transport, and secretion of glycoproteins (for review see Ref. 7). Upon arrival in the cis-Golgi, the initial mannose-rich core chains are trimmed by mannosidases I and II to continue N-glycosylation and O-linked glycosylation starts but may occur at multiple sites within the Golgi (8). In the medial-and trans-Golgi, the formation of complex type glycans ensues, which is terminated by the addition of sialic acid or other sugars. Sialyltransferases are responsible for the addition of sialic acid. Changes in the expression and enzyme activity of ST3Gal I and ST6Gal II, which catalyze the addition of sialic acid in positions ␣2,3 or ␣2,6, respectively, are associated with alterations in the length and structure of glycoproteins in several tumors and have been associated with malignant potential (9, 10). In melanoma cells, altered sialylation of surface glycoconjugates has been associ-
doi:10.1074/jbc.m608449200 pmid:17303571 fatcat:vkseq75wujcvtolkgobmsitsfi