Differential Processing and Secretion of the Melanoma-Associated Me20 Antigen

Maresh, Grace A.; Wang, W.C.; Beam, Katie; Malacko, Alison R.; Hellstrom, I.; Hellström, Karl Erik; Marquardt, Hans

doi:10.1006/abbi.1994.1213

Cited by 35 publications

(62 citation statements)

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“…2, a-e). As expected, secreted Pmel, which has been shown to correspond to M␣ (5,24), was immunoprecipitated by ␣Pmel-N, HMB50, and NKI-beteb, but not by the C terminus-directed ␣Pep13h (Fig. 2, a-e).…”

Section: Resultssupporting

confidence: 77%

“…Four Pmel products with luminal domains of 525, 532, 567, and 574 residues result from alternatively spliced mRNAs (21)(22)(23), with the 567-residue form predominating in most melanocytic cells. As for other type I integral membrane proteins, the signal sequence is cleaved, and at least four core N-linked oligosaccharides are added to consensus attachment sites, presumably cotranslationally, in the endoplasmic reticulum (ER) (5,24). At least a fraction of Pmel traverses the Golgi complex, where some of the N-linked oligosaccharides are modified by resident mannosidases and glysosyltransferases to a complex form that is resistant to digestion in vitro by endoglycosidase H (EndoH) (5,24) and where O-linked oligosaccharides are added, elaborated, and modified by sialic acid (25).…”

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confidence: 99%

“…As for other type I integral membrane proteins, the signal sequence is cleaved, and at least four core N-linked oligosaccharides are added to consensus attachment sites, presumably cotranslationally, in the endoplasmic reticulum (ER) (5,24). At least a fraction of Pmel traverses the Golgi complex, where some of the N-linked oligosaccharides are modified by resident mannosidases and glysosyltransferases to a complex form that is resistant to digestion in vitro by endoglycosidase H (EndoH) (5,24) and where O-linked oligosaccharides are added, elaborated, and modified by sialic acid (25). At least a fraction of the mature form is cleaved into two fragments, referred to here as M␣ and M␤, in a post-Golgi compartment (5) (likely endosomes; see Ref.…”

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confidence: 99%

“…26) by a proprotein convertase (15). A small fraction of the resulting luminal fragment is secreted (5,24). Pmel that accumulates in fibrillar stage II melanosomes is reactive with three commonly used antibodies, HMB45, HMB50, and NKI-beteb, all developed initially as melanoma markers (27).…”

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confidence: 99%

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Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

Harper

Theos

Herman

et al. 2008

Journal of Biological Chemistry

130

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Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.

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Section: Resultssupporting

confidence: 77%

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confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

Harper

Theos

Herman

et al. 2008

Journal of Biological Chemistry

130

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“…Those two proteins, known as Pmel17 and gp100, differ as to whether they include a 7-amino acid motif in the membrane proximal region of the luminal domain (1)(2)(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.…”

mentioning

confidence: 99%

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

Valencia

Rouzaud

Julien

et al. 2007

Journal of Biological Chemistry

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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.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 membrane 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, intrace...

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Study of Exosomes Shed New Light on Physiology of Amyloidogenesis

Niel

2016

Cell Mol Neurobiol

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Accumulation of toxic amyloid oligomers, a key feature in the pathogenesis of amyloid-related diseases, results from an imbalance between generation and clearance of amyloidogenic proteins. Cell biology has brought to light the key roles of multivesicular endosomes (MVEs) and their intraluminal vesicles (ILVs), which can be secreted as exosomes, in amyloid generation and clearance. To better understand these roles, we have investigated a relevant physiological model of amyloid formation in pigment cells. These cells have tuned their endosomes to optimize the formation of functional amyloid fibrils from the premelanosome protein (PMEL) and to avoid potential accumulation of toxic species. The functional amyloids derived from PMEL reveal striking analogies with the generation of Aβ peptides. We have recently strengthened these analogies using extracellular vesicles as reporters of the endosomal processes that regulate PMEL melanogenesis. We have shown that in pigmented cells, apolipoprotein E (ApoE) is associated with ILVs and exosomes, and regulates the formation of PMEL amyloid fibrils in endosomes. This process secures the generation of amyloid fibrils by exploiting ILVs as amyloid-nucleating platforms. This physiological model of amyloidogenesis could shed new light on the roles of MVEs and exosomes in conditions with pathological amyloid metabolism, such as Alzheimer's disease.

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Differential Processing and Secretion of the Melanoma-Associated Me20 Antigen

Cited by 35 publications

References 0 publications

Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

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

Study of Exosomes Shed New Light on Physiology of Amyloidogenesis

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