A Novel Splice Variant of Pmel17 Expressed by Human Melanocytes and Melanoma Cells Lacking Some of the Internal Repeats
2003; Elsevier BV; Volume: 121; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2003.12474.x
ISSN1523-1747
AutoresSarah Nichols, Dawn C. Harper, Joanne F. Berson, Michael S. Marks,
Tópico(s)Mast cells and histamine
ResumoPmel17 is a ≈100 kDa pigment cell specific glycoprotein that plays a crucial part in the morphogenesis of melanosome precursors. Anti-Pmel17 immunoprecipitates from metabolically pulse labeled melanoma cells and melanocytes contain, in addition to full-length Pmel17, a glycoprotein that migrates with a lower relative molecular weight. Here we show that this glycoprotein is encoded by an mRNA that results from alternative splicing of the human Pmel17 gene from which a cryptic intron is excised. Immunoprecipitation recapture experiments showed that this glycoprotein contained both the N- and C-termini of full-length Pmel17. Sequence analysis of cDNA corresponding to the alternatively spliced form reveals the loss of three of 10 imperfect direct repeats from the central region of the lumenal domain. The product of the splice variant is processed with similar kinetics to full-length Pmel17, and localizes similarly to late endosomes when expressed ectopically in nonpigment cells. We speculate that truncation of the repeat region within Pmel17 alters either fibrillogenic activity or the interaction of Pmel17 with melanin intermediates. The expression of an alternatively spliced product may furthermore affect the cohort of peptides generated for recognition of melanoma cells by tumor-directed T lymphocytes. Pmel17 is a ≈100 kDa pigment cell specific glycoprotein that plays a crucial part in the morphogenesis of melanosome precursors. Anti-Pmel17 immunoprecipitates from metabolically pulse labeled melanoma cells and melanocytes contain, in addition to full-length Pmel17, a glycoprotein that migrates with a lower relative molecular weight. Here we show that this glycoprotein is encoded by an mRNA that results from alternative splicing of the human Pmel17 gene from which a cryptic intron is excised. Immunoprecipitation recapture experiments showed that this glycoprotein contained both the N- and C-termini of full-length Pmel17. Sequence analysis of cDNA corresponding to the alternatively spliced form reveals the loss of three of 10 imperfect direct repeats from the central region of the lumenal domain. The product of the splice variant is processed with similar kinetics to full-length Pmel17, and localizes similarly to late endosomes when expressed ectopically in nonpigment cells. We speculate that truncation of the repeat region within Pmel17 alters either fibrillogenic activity or the interaction of Pmel17 with melanin intermediates. The expression of an alternatively spliced product may furthermore affect the cohort of peptides generated for recognition of melanoma cells by tumor-directed T lymphocytes. endoglycosidase H Melanin biosynthesis and storage are sequestered within lysosome-related organelles of melanocytes and eye pigment epithelia called melanosomes (Orlow, 1995Orlow S.J. Melanosomes are specialized members of the lysosomal lineage of organelles.J Invest Dermatol. 1995; 105: 3-7Crossref PubMed Scopus (225) Google Scholar; Marks and Seabra, 2001Marks M.S. Seabra M.C. The melanosome: Membrane dynamics in black and white.Nature Rev Mol Cell Biol. 2001; 2: 738-748Crossref PubMed Scopus (330) Google Scholar). In order to carry out their unique function, melanosomes harbor a cohort of glycoproteins that are expressed only in pigment cells. These proteins include enzymes, such as tyrosinase and the tyrosinase-related proteins Tyrp1 and Dopachrome tautomerase, as well as structural proteins and transporters that modulate the morphology, intralumenal microenvironment, and function of the melanosome (Sturm et al., 2001Sturm R.A. Teasdale R.D. Box N.F. Human pigmentation genes: Identification, structure and consequences of polymorphic variation.Gene. 2001; 277: 49-62Crossref PubMed Scopus (297) Google Scholar). Melanosome activity and morphology and the type of melanins produced can be modulated by the expression or modification of various components through gene regulatory mechanisms or mutation (Hearing, 1999Hearing V.J. Biochemical control of melanogenesis and melanosomal organization.J Invest Dermatol Symp Proc. 1999; 4: 24-28Abstract Full Text PDF PubMed Scopus (179) Google Scholar, Hearing, 2000Hearing V.J. The melanosome: The perfect model for cellular responses to the environment.Pigment Cell Res. 2000; 13: 23-34Crossref PubMed Scopus (102) Google Scholar). This kind of modulation can affect pigmentation in primary melanocytes or tumorigenicity and/or immunogenicity in transformed melanocytes (Kawakami et al., 2000Kawakami Y. Suzuki Y. Shofuda T. et al.T cell immune responses against melanoma and melanocytes in cancer and autoimmunity.Pigment Cell Res. 2000; 13: 163-169Crossref PubMed Scopus (37) Google Scholar; Overwijk and Restifo, 2000Overwijk W.W. Restifo N.P. Autoimmunity and the immunotherapy of cancer: Targeting the "self" to destroy the "other".Crit Rev Immunol. 2000; 20: 433-450Crossref PubMed Google Scholar; Slingluff et al., 2000Slingluff Jr, C.L. Colella T.A. Thompson L. et al.Melanomas with concordant loss of multiple melanocytic differentiation proteins: Immune escape that may be overcome by targeting unique or undefined antigens.Cancer Immunol Immunother. 2000; 48: 661-672Crossref PubMed Scopus (74) Google Scholar). Pmel17 (also known as gp100 or the product of the Silver locus) is a type I integral membrane glycoprotein that localizes to the lumen of melanosome precursors (Kwon et al., 1987Kwon B.S. Halaban R. Kim G.S. Usack L. Pomerantz S. Haq A.K. A melanocyte-specific complementary DNA clone whose expression is inducible by melanotropin and isobutylmethyl xanthine.Mol Biol Med. 1987; 4: 339-355PubMed Google Scholar; Orlow et al., 1993Orlow S.J. Zhou B.-K. Boissy R.E. Pifko-Hirst S. Identification of a mammalian melanosomal matrix glycoprotein.J Invest Dermatol. 1993; 101: 141-144Crossref PubMed Scopus (15) Google Scholar; Kobayashi et al., 1994Kobayashi T. Urabe K. Orlow S.J. et al.The Pmel17/silver locus protein. Characterization and investigation of its melanogenic function.J Biol Chem. 1994; 269: 29198-29205Abstract Full Text PDF PubMed Google Scholar; Lee et al., 1996Lee Z.H. Hou L. Moellmann G. et al.Characterization and subcellular localization of human Pmel17/silver, a 100-kDa (pre) melanosomal membrane protein associated with 5,6,-dihydroxyindole-2-carboxylic acid (DHICA) converting activity.J Invest Dermatol. 1996; 106: 605-610Crossref PubMed Scopus (70) Google Scholar). It is highly expressed by melanocytes, and serves as a common target for tumor-directed T lymphocytes in patients with melanoma (Kawakami et al., 1998aKawakami Y. Robbins P.F. Wang R.F. Parkhurst M. Kang X.Q. Rosenberg S.A. The use of melanosomal proteins in the immunotherapy of melanoma.J Immunother. 1998; 21: 237-246Crossref PubMed Scopus (75) Google Scholar). We have shown that Pmel17 aligns with the intralumenal fibers that are characteristic of melanosomes and their precursors (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar; Raposo et al., 2001Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.J Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (332) Google Scholar), that ectopic expression of Pmel17 alone is sufficient to induce the formation of similar fibers in nonpigment cells (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar), and that a lumenal domain fragment copurifies with the fibers (Berson et al., 2003Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis.J Cell Biol. 2003; 161: 521-523Crossref PubMed Scopus (214) Google Scholar). Others have implicated roles for Pmel17 in immobilizing the melanin intermediate, DHICA (Chakraborty et al., 1996Chakraborty A.K. Platt J.T. Kim K.K. Kwon B.S. Bennett D.C. Pawelek J.M. Polymerization of 5,6-dihydroxyindole-2-carboxylic acid to melanin by the Pmel 17/silver locus protein.Eur J Biochem. 1996; 236: 180-188Crossref PubMed Scopus (92) Google Scholar; Lee et al., 1996Lee Z.H. Hou L. Moellmann G. et al.Characterization and subcellular localization of human Pmel17/silver, a 100-kDa (pre) melanosomal membrane protein associated with 5,6,-dihydroxyindole-2-carboxylic acid (DHICA) converting activity.J Invest Dermatol. 1996; 106: 605-610Crossref PubMed Scopus (70) Google Scholar), and in detoxifying DHICA and/or other melanin intermediates within the melanocyte (Quevedo et al., 1981Quevedo W.C. Fleischmann R.D. Dyckman J. Premature loss of melanocytes from hair follicles of light (Blt) and silver (si) mice.in: Seiji M. Phenotypic Expression in Pigment Cells. Tokyo University Press, Tokyo1981: 177-184Google Scholar; Spanakis et al., 1992Spanakis E. Lamina P. Bennett D.C. Effects of the developmental colour mutations silver and recessive spotting on proliferation of diploid and immortal mouse melanocytes in culture.Development. 1992; 114: 675-680PubMed Google Scholar); both of these activities may be associated with the fibrillogenic activity of Pmel17, resulting in immobilization of melanin intermediates on fibrous structures. The hypopigmentation of silver mice, which harbor a truncated Pmel17 molecule (Martínez-Esparza et al., 1999Martínez-Esparza M. Jiménez-Cervantes C. Bennett D.C. Lozano J.A. Solano F. García-Borrón J.C. The mouse silver locus encodes a single transcript truncated by the silver mutation.Mammalian Genome. 1999; 10: 1168-1171Crossref PubMed Scopus (44) Google Scholar), indicates that an intact cytoplasmic domain is required for Pmel17 function. The structural features of the Pmel17 lumenal domain that effect function within melanosomes, however, are not known. During its biosynthesis within melanocytes, Pmel17 undergoes complex processing, involving cleavage within the lumenal domain, which is required for its fibrillogenic activity (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar), suggesting that its function may require conformational changes induced by proteolytic processing of this domain. The only other recognizable features of the lumenal domain are (1) a small region with homology to polycystic kidney disease (PKD) repeat regions within the PKD 1 protein (Hughes et al., 1995Hughes J. Ward C.J. Peral B. et al.The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains.Nature Genet. 1995; 10: 151-160Crossref PubMed Scopus (720) Google Scholar), and (2) 10 tandem repeats of a 13 amino acid proline- and glutamate-rich sequence immediately downstream of the PKD repeats (Kwon et al., 1991Kwon B.S. Chintamaneni C. Kozak C.A. et al.A melanocyte-specific gene, Pmel17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12.Proc Natl Acad Sci USA. 1991; 88: 9228-9232Crossref PubMed Scopus (130) Google Scholar). Two distinct Pmel17 products resulting from alternative splicing of the mRNA have been described in human melanocytic cells (Adema et al., 1994Adema G.J. de Boer A.J. Vogel A.M. Loenen W.A.M. Figdor C.G. Molecular characterization of the melanocyte lineage-specific antigen gp100.J Biol Chem. 1994; 269: 20126-20133Abstract Full Text PDF PubMed Google Scholar; Maresh et al., 1994Maresh G.A. Marken J.S. Neubauer M. Aruffo A. Hellström I. Hellström K.E. Marquardt H. Cloning and expression of the gene for the melanoma-associated ME20 antigen.DNA Cell Biol. 1994; 13: 87-95Crossref PubMed Scopus (27) Google Scholar; Bailin et al., 1996Bailin T. Lee S.T. Spritz R.A. Genomic organization and sequence of D12S53E (Pmel 17), the human homologue of the mouse silver (si) locus.J Invest Dermatol. 1996; 106: 24-27Crossref PubMed Scopus (17) Google Scholar; Kim et al., 1996Kim K.K. Youn B.S. Heng H.H. et al.Genomic organization and FISH mapping of human Pmel17, the putative silver locus.Pigment Cell Res. 1996; 9: 42-48Crossref PubMed Scopus (9) Google Scholar). These two forms differ by only seven amino acids, encoded by a 21 bp insertion resulting from use of an alternative splice acceptor site in exon 10 of the Pmel17 gene, within the juxtamembrane region of the lumenal domain (Bailin et al., 1996Bailin T. Lee S.T. Spritz R.A. Genomic organization and sequence of D12S53E (Pmel 17), the human homologue of the mouse silver (si) locus.J Invest Dermatol. 1996; 106: 24-27Crossref PubMed Scopus (17) Google Scholar; Kim et al., 1996Kim K.K. Youn B.S. Heng H.H. et al.Genomic organization and FISH mapping of human Pmel17, the putative silver locus.Pigment Cell Res. 1996; 9: 42-48Crossref PubMed Scopus (9) Google Scholar). Here, we describe a second splice variation that occurs in human melanocytic cells and results in the production of a smaller Pmel17 product containing a deletion within the lumenal domain tandem repeat region. We speculate that expression of this truncated Pmel17 may alter oligomerization or melanin binding properties and thereby modulate melanosome structure and/or function. Human melanoma cell lines MNT-1 and 1011-mel were cultured in Dulbecco minimal Eagle's medium supplemented with 10% AIM-V medium, 20% fetal bovine serum, 1 mM sodium pyruvate, nonessential amino acids, and antibiotics (penicillin and streptomycin). Melan-a (Bennett et al., 1989Bennett D.C. Cooper P.J. Dexter T.J. Devlin L.M. Heasman J. Nester B. Cloned mouse melanocyte lines carrying the germline mutations albino and brown: Complementation in culture.Development. 1989; 105: 379-385Crossref PubMed Google Scholar) cells and the mouse T cell hybridoma, DO11.10 (Roehm et al., 1982Roehm N.W. Marrack P. Kappler J.W. Antigen-specific, H-2-restricted helper T cell hybridomas.J Exp Med. 1982; 156: 191-204Crossref PubMed Scopus (33) Google Scholar) were cultured as described. Primary melanocytes, a kind gift from Dr M. Herlyn and R. Finko, were cultured as described (Raposo et al., 2001Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.J Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (332) Google Scholar). HeLa cells were cultured as described (Calvo et al., 1999Calvo P.A. Frank D.W. Bieler B.M. Berson J.F. Marks M.S. A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery.J Biol Chem. 1999; 274: 12780-12789Crossref PubMed Scopus (96) Google Scholar), and transfected using FuGene-6 (Roche Applied Science, Indianapolis, Indiana) according to the manufacturer's instructions with 2 μg of total plasmid DNA (0.2 μg of specific plasmid DNA and 1.8 μg of empty pCI expression vector). αPmel-N and αPmel-I antibodies were raised against peptides corresponding to N-terminal or internal regions of the Pmel17 lumenal domain. Peptides were synthesized with the following sequences, representing the indicated residues from full-length Pmel17 with an additional N-terminal cysteine added for coupling to keyhole limpet hemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide ester: Pmel-N, H2N-(C)TKVPRNQDWLGVSRQLR-CO2H (residues 24–40); Pmel-I, H2N-(C)QVPTTEVVGTTPGQAPTAE-CO2H (residues 326–344). Peptide synthesis, coupling, rabbit immunization and production were performed by Genemed Synthesis (South San Francisco, California). Anti-peptide antibodies ere affinity purified by passing sodium sulfate precipitated serum through a column in which peptides were coupled to SulfoLink gel (Pierce Biotechnology, Rockford, Illinois) according to the manufacturer's instructions. Antibodies were eluted with 0.1 M glycine pH 2.7, neutralized with Tris base, and dialyzed in phosphate-buffered saline containing 0.02% sodium azide. Other antibodies and their sources were as follows: αPEP13h (referred to here as αPmel-C for simplification), to Pmel17 cytoplasmic domain (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar); HMB50 and HMB45, to Pmel17 lumenal domain (Lab Vision, Fremont, California); and H4A3, to LAMP1 (Developmental Studies Hybridoma Bank, Iowa City, Iowa). Chromophore-conjugated secondary antibodies were obtained from Jackson Immunoresearch (West Grove, Pennsylvania) or from Southern Biotech (Birmingham, Alabama). mRNA was isolated from MNT-1, 1011-mel, HeLa, melan-a, cultured primary human melanocytes, or D011.10 mouse T cell hybridoma cells using the RNEasy kit (Qiagen, Valencia, California). For reverse transcriptase reactions, RNA (15 μg) was incubated with reverse primers, as indicated, heated to 90°C, cooled to 52°C for 15 min, and then incubated with Avian Myeloblastosis Virus reverse transcriptase and appropriate buffers according to the manufacturer's instructions (Promega, Madison, Wisconsin). Control reactions lacked reverse transcriptase. After ethanol precipitation, 12.5% of each reaction was subjected to PCR amplification with indicated primers for 30 cycles (1 min at 95°C, 1 min at 55°C, and 2.5 min at 72°C). Parallel reactions were done using 100 ng of plasmid DNA with Pmel17-l insert. Reaction products were fractionated by agarose gel electrophoresis alongside 100 bp markers (Life Technologies, Rockville, Maryland) and visualized after staining with ethidium bromide. Triads of PCR reactions with template from reverse transcriptase reactions that contained or lacked reverse transcriptase enzyme or with plasmid template are shown at identical exposures for each triad. Primers are indicated in Table I.Table IOligonucleotides used for reverse transcription–PCRNo.DerivationPosition in cDNAaReverse primers are indicated by (R).Sequence of oligonucleotidebSuperfluous sequences that are not directly homologous to the cDNA are indicated by italics.144hPmel17451–474457GCA TCT TCC CTG ATG GTG474145hPmel17855–872855GGA GAC AGT AGT GGA ACC872147hPmel171654–16701654CAT CGC CAG GGT GCC AG1670166hPmel171938–1952GGG CTC ACC TGG CAA1938AGG CGC AGA CTT ATG1952171hPmel171454–14921454C ACC TTA AGG CTG GTG CAG CAA CAA GTC CCC CTG GAT TG1492172hPmel171492–1454 (R)1492CA ATC CAG GGG GAC TTG TTG CTG CAC CAG CCT TAA GGT G1454356hPmel171998–1983 (R)tc ttt tct aga tt1998A GTG ACT GCT GCT ATG1983398hPmel172119–2102 (R)tgt ttt cta ga2119g ttt ctg tca act cca gg2102232mGAPDH47–6947ATG GTG AAG GTC GGT GTG AAC GG69233mGAPDH1045–1018 (R)1045CTC CTT GGA GGC CAT GTA GGC CAT CAG G1018319hRab5a57–73atac cag atc ttg57ATG GCT AGT CGA GGC GC73321hRab5a704–682 (R)aaga tct aga704TTA GTT ACT ACA ACA CTG ATT CC682418mPmel17902–921902GT GGT TCC TCC CCA GTC CCG921419mPmel171320–1302 (R)1320CAG GGG AAC TTG TCT CTT C1302a Reverse primers are indicated by (R).b Superfluous sequences that are not directly homologous to the cDNA are indicated by italics. Open table in a new tab For sequencing and subcloning, PCR reaction products using primers 144 and 398 were fractionated by agarose gel electrophoresis, purified, and subcloned into pCR2.1 by TA cloning (Invitrogen, Carlsbad, California). Clones with appropriate sized EcoRI inserts for Pmel17-s and Pmel17-i were sequenced by automated dideoxy sequence analysis (Children's Hospital, Philadelphia, Pennsylvania). To reconstitute a full-length cDNA for hPmel17-s, the NcoI–XbaI fragment from pCR2.1-hPmel17-s was used to replace the corresponding fragment in full-length hPmel17-l subcloned in pSP72 (Promega). Similarly, full-length hPmel17-i was generated by using the BstXI–XbaI fragment from pCR2.1-hPmel17-i to replace the corresponding fragment in pSP72-hPmel17-l. From the resultant pSP72 plasmids containing full-length products, EcoRI–XbaI fragments containing the entire reading frame of hPmel17-s or hPmel17-i were subcloned into the pCI mammalian expression vector (Promega). Sequences of all inserts were verified by automated dideoxy sequencing. Melanoma cells, melanocytes, or transfected HeLa cells were metabolically labeled with 35S-methionine/cysteine as described (Berson et al., 2000Berson J.F. Frank D.W. Calvo P.A. Bieler B.M. Marks M.S. A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature.J Biol Chem. 2000; 275: 12281-12289Crossref PubMed Scopus (104) Google Scholar) for 20 to 30 min, then chased for 0 to 4 h as indicated. Cell lysates in 1% Triton X-100 were prepared and immunoprecipitated as described (Berson et al., 2000Berson J.F. Frank D.W. Calvo P.A. Bieler B.M. Marks M.S. A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature.J Biol Chem. 2000; 275: 12281-12289Crossref PubMed Scopus (104) Google Scholar). For immunoprecipitation/recapture experiments, immunoprecipitated products were released by addition of 50 μL of 0.5% sodium dodecyl sulfate (SDS) and heating to 100°C for 5 min. Samples were then brought to 0.5 mL with lysis buffer to dilute the SDS, and subjected to a second round of immunoprecipitation with indicated antibodies. All immunoprecipitates were fractionated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed on a Molecular Dynamics Storm Phosphor Imaging system using ImageQuant software (Amersham Biosciences, Piscataway, New Jersey). Whole cell lysates of transfected HeLa cells were treated or not with endoglycosidase H (EndoH, New England Biolabs, Beverly, MA), fractionated by SDS–PAGE, transferred to reinforced nitrocellulose membranes, probed with αPmel-N or αPmel-C antibodies, and developed with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin and ECF enhanced chemiluminescence (Amersham Biosciences) as described previously (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar). Transiently transfected HeLa cells expressing hPmel17-l, hPmel17-i, or hPmel17-s were fixed with 2% formaldehyde in phosphate-buffered saline and stained as described (Calvo et al., 1999Calvo P.A. Frank D.W. Bieler B.M. Berson J.F. Marks M.S. A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery.J Biol Chem. 1999; 274: 12780-12789Crossref PubMed Scopus (96) Google Scholar) with antibodies to Pmel17 (HMB50; IgG2a) and to Lamp1 (H4A3; IgG1), followed by isotype-specific secondary antibodies conjugated to fluorescein isothiocyanate or Texas Red. Cells were analyzed on a Leica Microsystems (Bannockburn, Illinois) DM IRBE microscope, and images were captured, analyzed, and processed for deconvolution using a Hamamatsu (Hamamatsu, Japan) Orca digital camera and Improvision (Lexington, Massachusetts) OpenLab software. Sequence formatting and comparisons were done using DNAStar (Madison, Wisconsin) or DNA Strider. The following GenBank accession nos were used to obtain cDNA sequences for Pmel17 orthologs from the NCBI database: mouse (Mus musculus) Silver, accession no. NM_021882; chicken (Gallus gallus) MMP115, accession no. D88348; bovine (Bos taurus) RPE1, accession no. M81193; and horse (Equus caballus) Pmel17, accession no. AF076780. Anti-Pmel17 immunoprecipitates of cell lysates from metabolically pulse labeled MNT-1 human melanoma cells contained, in addition to the ≈100 kDa band representing full-length Pmel17, a band with faster migration (≈95 kDa;Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar). This band, referred to as "band X" was: (1) precipitable with either of two distinct antibodies to Pmel17 (HMB50, which recognizes the lumenal domain, and αPmel-C, which binds to the cytoplasmic domain), (2) susceptible to digestion with EndoH (indicating residence within a pre-Golgi compartment), and (3) not obvious in lysates from cells that were chased in the presence of excess unlabeled methionine (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar; see alsoBerson et al., 2003Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis.J Cell Biol. 2003; 161: 521-523Crossref PubMed Scopus (214) Google Scholar). The band was distinguished from the proteolytic product, Mα, by virtue of its slightly distinct migration in gels and by its susceptibility to digestion with EndoH; Mα, which appears only after the chase, is resistant to EndoH digestion. Furthermore, band X was not observed in lysates of transfected HeLa cells expressing Pmel17 from a transgene (Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar). To determine whether band X was unique to MNT-1 cells or represented a coprecipitating product present in untransformed cells, we subjected cultures of normal primary human melanocytes to a similar metabolic pulse/chase analysis. As shown in Figure 1, a similar pattern of immunoprecipitable products was observed from Triton X-100 cell lysates of these cells as previously observed in MNT-1 cells. Thus, band X was present in addition to full-length, core glycosylated "P1" Pmel17 at the pulse. During the chase, both were reduced in intensity and a slightly faster migrating Mα band appeared concomitantly with the ≈28 kDa Mβ and the faint, diffuse slower migrating P2 form representing the Golgi modified full-length protein (note that unlike P1 and band X, the migration of Mα and P2 varies among melanoma cells and primary melanocytes most likely due to differential terminal glycosylation;Vogel and Esclamado, 1988Vogel A.M. Esclamado R.M. Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody.Cancer Res. 1988; 48: 1286-1294PubMed Google Scholar; Chiamenti et al., 1996Chiamenti A.M. Vella F. Bonetti F. et al.Anti-melanoma monoclonal antibody HMB-45 on enhanced chemiluminescence-western blotting recognizes a 30–35 kDa melanosome-associated sialated glycoprotein.Melanoma Res. 1996; 6: 291-298Crossref PubMed Scopus (35) Google Scholar). Although reduced in intensity, P1 persists throughout the chase in all cells, likely due to inefficient exit from the endoplasmic reticulum (Vogel and Esclamado, 1988Vogel A.M. Esclamado R.M. Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody.Cancer Res. 1988; 48: 1286-1294PubMed Google Scholar; Adema et al., 1994Adema G.J. de Boer A.J. Vogel A.M. Loenen W.A.M. Figdor C.G. Molecular characterization of the melanocyte lineage-specific antigen gp100.J Biol Chem. 1994; 269: 20126-20133Abstract Full Text PDF PubMed Google Scholar; Kobayashi et al., 1994Kobayashi T. Urabe K. Orlow S.J. et al.The Pmel17/silver locus protein. Characterization and investigation of its melanogenic function.J Biol Chem. 1994; 269: 29198-29205Abstract Full Text PDF PubMed Google Scholar; Berson et al., 2001Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Pmel17 initiates premelanosome morphogenesis within multivesicular bodies.Mol Biol Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (247) Google Scholar, Berson et al., 2003Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis.J Cell Biol. 2003; 161: 521-523Crossref PubMed Scopus (214) Google Scholar). All bands were precipitable with both HMB50 and αPmel-C, indicating that lumenal and cytoplasmic domains were both present (and linked in the case of Mα/Mβ). Similar results were obtained in a second primary human melanocyte culture and in 1011-mel human melanoma cells. By contrast, only one band corresponding to the full-length product was observed in immunoprecipitates of pulse-labeled mouse melanocytic cell lines melan-a and melan-a3 (unpublished data). Thus, band X represents a coprecipitating product present in normal and transformed human melanocytes but absent in mouse melanocytes. To determine whether band X represented a modified form of Pmel17 or a coprecipitating product, we subjected radio-labeled cell lysates to a two-step immunoprecipitation recapture protocol using anti-Pmel17 antibodies. Pmel17 was first immuno-precipitated using αPmel-C or HMB50 under nondenaturing conditions from Triton X-100 lysates of 35S-methionine pulse-labeled MNT-1 cells (Figure 2a, lanes 1 and 2) or cells chased for 2 h in the absence of 35S-methionine (Figure 2b, lanes 12 and 13). Material from these primary immunoprecipitates was then eluted by boiling in the presence of SDS and a reducing agent; this treatment not only dissociates material from the antibodies, but al
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