Translation Rate of Human Tyrosinase Determines ItsN-Linked Glycosylation Level

Abstract

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or sevenN-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2–3-fold faster (7.7–10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinaseN-linked glycosylation, as well as other potential cotranslational maturation events. Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or sevenN-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2–3-fold faster (7.7–10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinaseN-linked glycosylation, as well as other potential cotranslational maturation events. endoplasmic reticulum azetidine-2-carboxylic acid cycloheximide deoxynojirimycin green fluorescent protein murine major histocompatibility complex class I molecule Kbsignal sequence oligosaccharyl transferase polyacrylamide gel electrophoresis rough ER rabbit reticulocyte lysate human tyrosinase tyrosinase with 6 glycans tyrosinase with 7 glycans untranslocated tyrosinase wheat germ 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Whereas prokaryotic proteins fold posttranslationally due to their rapid rate of translation, the maturation of nascent proteins in eukaryotic cells often begins cotranslationally as a vectorial process and continues posttranslationally after the release of the protein from the ribosome (1Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. 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The biosynthesis of melanin takes place in post-Golgi endomembranous compartments called melanosomes or pigmented granules. Mutational analysis of tyrosinase-positive albinism has identified AP-3 as an important protein involved in the sorting of tyrosinase in thetrans-Golgi to melanosomes (26–28, reviewed in Ref. 29Spritz R.A. Trends Genet. 1999; 15: 337-340Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, additional key sorting decisions for tyrosinase are made in the early secretory pathway that are critical for pigmentation (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar, 31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar, 32Petrescu S.M. Branza-Nichita N. Negroiu G. Petrescu A.J. Dwek R.A. Biochemistry. 2000; 39: 5229-5237Crossref PubMed Scopus (46) Google Scholar). Tyrosinase is a membrane glycoprotein with an N-terminal signal sequence that targets the protein to the ER (Fig. 1 A) (33Kwon B.S. Haq A.K. Pomerantz S.H. Halaban R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7473-7477Crossref PubMed Scopus (396) Google Scholar, 34Ruppert S. Muller G. Kwon B. Schutz G. EMBO J. 1988; 7: 2715-2722Crossref PubMed Scopus (141) Google Scholar, 35Bouchard B. Fuller B.B. Vijayasaradhi S. Houghton A.N. J. Exp. Med. 1989; 169: 2029-2042Crossref PubMed Scopus (158) Google Scholar, 36Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Jpn. J. Genet. 1989; 64: 121-135Crossref PubMed Scopus (94) Google Scholar). The human protein possesses 7 putative N-linked glycosylation sites and 15 lumenal Cys residues that can participate in disulfide bond formation. Mutations in tyrosinase are the cause of tyrosinase-negative oculocutaneous albinism 1, an autosomal recessive genetic disorder characterized by the absence of melanin (reviewed in Refs. 37Oetting W.S. King R.A. Hum. Mutat. 1999; 13: 99-115Crossref PubMed Scopus (280) Google Scholar and 38King R.A. Nordlund J.J. Boissy R. Hearing V.J. King R.A. Ortonne J.-P. The Pigmentary System. Physiology and Pathophysiology. 1st Ed. Oxford University Press, New York1998Google Scholar). The mutant protein in several tyrosinase-negative albino melanocytes of human and mouse origin is retained in the ER (31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar,39Berson J.F. Frank D.W. Calvo P.A. Bieler B.M. Marks M.S. J. Biol. Chem. 2000; 275: 12281-12289Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The essential role of the ER in the regulation of tyrosinase has also been demonstrated in amelanotic melanoma cells in which ER retention of wild type tyrosinase leads to subsequent degradation by the 26 S proteasome (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). Here, we demonstrate that the faster rate of translation of tyrosinase in melanoma cells than that of normal melanocytes hampered glycosylation at the inefficient Asn-290 site. This was determined by studying tyrosinase glycosylation and maturation under conditions that altered the rate of translation in normal melanocytes and melanoma cells, as well as in a cell-free system that recapitulated the ER processes. Rabbit reticulocyte lysate (RRL), wheat germ (WG), dithiothreitol, and RNasin were from Promega Corp. (Madison, WI). Canine pancreas microsomes were a generous gift from Dr. R. Gilmore (Worcester, MA). [35S]Methionine/cysteine (EasyTag) and CHAPS were from PerkinElmer Life Sciences and Pierce, respectively. Restriction endonucleases and ribonucleotide triphosphates were from New England Biolabs, Inc. (Beverly, MA). mMessage mMachine and T7 transcription kits were from Ambion (Austin, TX), and the QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Zysorbin (fixed and killedStaphylococcus aureus) was obtained from Zymed Laboratories Inc. (San Francisco, CA). All other reagents, including the anti-FLAG M2 monoclonal antibody, were from Sigma. The plasmid pcTYR carrying the human tyrosinase gene (GenBankTM accession number Y00819) was a gift from Dr. R. Spritz (Denver, CO). The EcoRI tyrosinase fragment excised from pcTYR was subcloned into pGEM 7Zf (Promega). To improvein vitro translation/translocation, the original signal sequence of tyrosinase was exchanged with the murine major histocompatibility complex class I molecule Kbsignal sequence as follows. A XbaI restriction site was introduced at the end of the 18-amino acid signal sequence in pGEM 7Zf-TYR plasmid by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene). The tyrosinase XbaI fragment (encoding the full sequence of tyrosinase minus the signal peptide) was inserted into the XbaI restriction site of pSP72/KbSS-CD3δ plasmid (from Drs. J. Huppa and H. Ploegh, Boston, MA) (40Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar). This created a hybrid molecule comprising the class I heavy chain H2-Kb signal peptide in frame with the tyrosinase protein downstream of the T7 promoter, termed pSP72/KbSS-TYR. To generate single-site glycosylation deletion mutant proteins, the consensus N-linked glycosylation sites Asn-X-Thr/Ser in pSP72/KbSS-TYR were eliminated or modified in the cDNA by changing threonine or serine to an alanine, except for Thr-373, which was changed to the albino mutation T373K. The site at Asn-290 was modified by exchanging the proline at position 293 to alanine (P293A). The P293A and T292A mutations were also introduced to tyrosinase cDNA in the enhanced green fluorescent protein (enhanced GFP) vector (31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar). In addition, the wild type and mutant cDNAs were subcloned from the enhanced GFP plasmids into theKpnI/HindII cloning sites of p3XFLAG-CMV-14 expression vector (Sigma) to generate FLAG-tagged tyrosinase proteins. In all cases, DNA sequencing of the entire tyrosinase gene verified the inserted mutations. Transfection of plasmids into mouse melanocytes was done as described (31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar). Messenger RNA was prepared by in vitro run-off transcription of the cDNA that was linearized with NdeI or HaeII, according to the manufacturer's instructions. Radioactive35S-labeled tyrosinase was translated for 1 h at 27 °C in RRL (14Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar) or in WG and translocated cotranslationally into canine pancreas microsomes. In the latter case, WG lysate (40 μl) was mixed with ribonuclease-treated rough ER (RER) microsomes (6 μl), an amino acid mixture lacking methionine and cysteine (6 μl of a 1 mm solution of each), [35S]Met/Cys (63 μCi), RNase-free water (18.4 μl), RNase inhibitor (1.6 μl), and mRNA (1 μg/μl, 3.5 μl). Samples were alkylated withN-ethylmaleimide (20 mm) to block free sulfhydryls (7Braakman I. Hoover-Litty H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (252) Google Scholar). Alkylated samples were either analyzed directly by SDS-PAGE or immunoprecipitated with anti-tyrosinase antibodies (α-TYR) prior to electrophoresis. Deoxynojirimycin (DNJ) (0.5 mm) was used to inhibit ER glucosidases when indicated. Half of each sample was subjected to nonreducing SDS-PAGE, and the other half was reduced by the addition of dithiothreitol (100 mm) and resolved by reducing SDS-PAGE. Semipermeabilized cells were prepared from subconfluent mouse B10BR melanocytes (41Bennett D.C. Cooper P.J. Dexter T.J. Devlin L.M. Heasman J. Nester B. Development. 1989; 105: 379-385Crossref PubMed Google Scholar) permeabilized with 20 μg/ml digitonin using a method described previously by Wilson et al. (42Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J. 1995; 307: 679-687Crossref PubMed Scopus (133) Google Scholar). Radioactive35S-labeled tyrosinase was translated for 1 h at 27 °C in RRL with semipermeabilized cells (1.3 × 104 cells/μl) replacing the RER microsomes. To separate glycosylated (TYR) from nonglycosylated (untranslocated (UTYR)) proteins, 10 μl of the translation mixture was solubilized in 200 μl of 2% CHAPS buffer (2% CHAPS, 50 mm HEPES, 200 mm sodium chloride, pH 7.5), and glycoproteins were captured on wheat germ agglutinin-bound beads at 4 °C for 2 h under constant rotation. The beads were pelleted by centrifugation at 2500 × g and washed once with 0.5% CHAPS buffer. The bound proteins were then eluted in SDS-sample buffer at 95 °C for 5 min. Alternatively, untranslocated tyrosinase was separated from the translocated protein by centrifugation of the translation products (10 μl) through a sucrose cushion (100 μl, 0.5m sucrose, 50 mm triethanolamine, 1 mm dithiothreitol, pH 7.4) in a Beckman Airfuge ultracentrifuge for 10 min. For protease protection, translation products were digested with proteinase K (0.35 μg/μl) in the absence or presence of 1% Triton X-100 for 1 h on ice. Protease digestion was stopped with 10 mm phenylmethylsulfonyl fluoride. Samples were added to a 100-μl solution of 0.1m Tris, 1% SDS (pH 8) and heated at 95 °C for 10 min. Translation mixtures were centrifuged through a sucrose cushion, and pellets were solubilized in a solution containing 0.2% SDS, 100 mm sodium phosphate, 25 mm EDTA (pH 6.9) at 95 °C for 5 min. The samples were cooled to room temperature, diluted with 2% Triton X-100 in 100 mm sodium phosphate, 25 mm EDTA (pH 6.9), and digested with 0.1–1 μl of 1 units/μl PNGase F at 37 °C for the indicated times. 35S-Labeled tyrosinase was immunoprecipitated with anti-tyrosinase antibodies (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). To determine radioactivity in total proteins, lysates were blotted on filter paper and treated with 10% trichloroacetic acid on ice for 30 min. Samples were washed with The radioactivity of the was determined in a The of in the in vitro translation was determined as described samples with [35S]Met/Cys were fully resolved by SDS-PAGE to the of the translocated tyrosinase by samples were also on the gel after the was Therefore, the samples were resolved and the of the total free in the Because tyrosinase has a total of and its is the × the of the tyrosinase and F is the of the total free is the of the of reticulocyte lysate and the [35S]Met/Cys human melanocytes were from in with and several for their including and M. Moellmann G. Cheng E. M. Wagner S. P. Halaban R. Cell 1995; 6: Google Scholar). cells and were in (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). were for 2 h in with 1% and 50 μg/ml were then with [35S]Met/Cys for 10 into with 20 mm and in 2% CHAPS buffer. Cell were immunoprecipitated with anti-tyrosinase antibodies (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). tyrosinase from normal melanocytes as a doublet separated by (Fig. 1 (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). doublet of similar also in melanoma cells, the of the individual doublet The of the to the in melanoma cells was half of that in normal melanocytes compared with that the faster in the melanoma cells. To the cause of the was in a cell-free system that The translation system of rabbit reticulocyte lysate and mRNA human in the presence and absence of RER (Fig. The native tyrosinase mRNA or a modified termed in which the signal sequence was exchanged with the signal sequence from murine major histocompatibility complex class I molecule (40Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar, J.J. Ploegh H. J. 1993; PubMed Scopus Google was A by anti-tyrosinase was in the absence of microsomes that to untranslocated and (Fig. 1 and However, a doublet to that observed previously in melanocytes (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar) (Fig. 1 in the presence of RER microsomes. This translocated tyrosinase that N-linked (Fig. 2 and The was translated and translocated than native tyrosinase (Fig. 2 to The untranslocated slower than wild type 1 to 2 5 to due to the of signal sequence amino acids in and amino acids in However, the of the translocated products was (Fig. 2 to 4 and 7 and was similar to tyrosinase in melanocytes, the of the in all subsequent cell-free The protein doublet the translocated and glycosylated tyrosinase it was with wheat germ agglutinin-bound beads (Fig. 2 and it through a sucrose cushion to with (Fig. 2 with proteinase K the the microsomes were solubilized by The by proteinase K cleavage of the acid cytosolic of the translocated is with correct of the type I membrane protein into the membrane 6 to the the that the in vitro translated doublet ER translocated and glycosylated of tyrosinase. The translocated ER of tyrosinase by glucose or of N-linked glycans in the ER or through by the generating in the total number of glycans. of glucose residues as the for this was by the production of doublet protein in the presence of the translated and products slower due to of glucosidases I and II and ER was by the of the doublet in the presence of the inhibitor in the side chains as the of doublet was indicated after digestion with the PNGase This side chains the doublet into a that faster than the untranslocated 6 and The faster of the PNGase F digested the untranslocated of tyrosinase 1 and 2 6 and was due to the cleavage of the signal sequence during RER Therefore, we that the doublet tyrosinase with ofN-linked glycans. The number of glycans was then resolved by a of PNGase F products (Fig. Tyrosinase was in the presence of to due to glucose digestion with of PNGase F an protein (Fig. However, PNGase F at a of to proteins with seven to glycans (Fig. This analysis revealed that the doublet tyrosinase with seven and six N-linked glycans. The digestion by PNGase F that the doublet was by inefficient glycosylation of of the consensus The the of inefficient glycosylation or of of the of a consensus site by inefficient transfer of with disulfide bond or the presence of inefficient sites B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. von Heijne G. 3: PubMed Scopus Google Scholar, S. Bulleid N.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J.R. EMBO J. 1996; 15: PubMed Scopus Google Scholar). The of was glycosylation of a of substrates has been with this cell-free system (14Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, Hebert D.N. Helenius A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, D.N. Zhang W. Foellmer B. Helenius A. J. Cell Biol. 1997; PubMed Scopus Google and the doublet in cells. we by disulfide bond the doublet when tyrosinase was in the presence of the reducing dithiothreitol (Fig. 2 and A that a proline a role in the from using the proline