Sialylated core 1 O-glycans influence the sorting of Pmel17/gp100 and determine its capacity to form fibrils.
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.
Valencia
Julio C
JC
Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Rouzaud
Francois
F
Julien
Sylvain
S
Chen
Kevin G
KG
Passeron
Thierry
T
Yamaguchi
Yuji
Y
Abu-Asab
Mones
M
Tsokos
Maria
M
Costin
Gertrude E
GE
Yamaguchi
Hiroshi
H
Jenkins
Lisa M Miller
LM
Nagashima
Kunio
K
Appella
Ettore
E
Hearing
Vincent J
VJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D045744:Cell Line, Tumor; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D020460:Melanosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D021381:Protein Transport; D013997:Time Factors; D058951:gp100 Melanoma Antigen
D000906:Antibodies; D008562:Membrane Glycoproteins; C548637:PMEL protein, human; D011134:Polysaccharides; D058951:gp100 Melanoma Antigen; D019158:N-Acetylneuraminic Acid