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The Molecular Basis of Chemical Chaperone Therapy for Oculocutaneous Albinism Type 1A
ORIGINAL ARTICLE
The Molecular Basis of Chemical Chaperone Therapy for Oculocutaneous Albinism Type 1A Ayako Teramae1, Yui Kobayashi1, Hiroyuki Kunimoto2, Koichi Nakajima2, Tamio Suzuki3, Daisuke Tsuruta1 and Kazuyoshi Fukai4 Oculocutaneous albinism (OCA) is an autosomal recessive disease characterized by the reduction or complete lack of melanin pigment in the skin, hair, and eyes. No effective treatment for OCA is available at present. OCA type 1 is caused by mutations that disrupt the function of tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis. Recently, it was shown that tyrosinase in some patients with OCA type 1 mutation is retained in the endoplasmic reticulum and that its catalytic activity is lost, a phenomenon known as endoplasmic reticulum retention. However, to our knowledge, the intracellular localization of tyrosinase in Japanese patients with OCA type 1 missense mutations has not been reported. In this study, we first investigated the intracellular localization of Japanese OCA type 1A missense mutant tyrosinases using Western blotting and immunohistochemical staining. R77Q, R239W, D383N, and P431L mutant tyrosinases were retained in the endoplasmic reticulum, and H211Y mutant tyrosinase was partially transported to the Golgi apparatus. Second, we explored the possibility of chemical chaperone therapy for Japanese patients with OCA type 1A missense mutations and found that HeLa cells expressing P431L mutant tyrosinase have restored tyrosinase activity after treatment with a low-dose tyrosinase inhibitor, as a chemical chaperone, in a dose-dependent manner. These results provide the basis for a possible chemical chaperone therapy to recover tyrosinase activities in patients with OCA type 1A patients. Journal of Investigative Dermatology (2018) -, -e-; doi:10.1016/j.jid.2018.10.033
INTRODUCTION Oculocutaneous albinism (OCA) is a group of autosomal recessive diseases of pigmentation caused by the complete lack or reduction of melanin biosynthesis in melanocytes. Seven types of OCA have been classified according to the genes responsible. Among them, OCA type 1 (OCA1) is characterized by hypopigmentation of the skin, hair, and eyes. OCA1 is caused by mutations in the tyrosinase gene (TYR). Tyrosinase is a copper-containing enzyme protein that is critically involved in the biosynthesis of melanin. Human tyrosinase is a type 1 membrane glycoprotein that contains 529 amino acids with a N-terminal signal sequence and a C-terminal transmembrane domain. It has seven asparagine-linked (N-linked) glycosylation sites (Wang and Hebert, 2006) and two copper binding sites, CuA and CuB. Those two sites each contain three histidine residues that coordinate the binding of copper (Lerner et al., 1950), CuA at His 180, 202, and 211 and CuB at His 363, 367, and 389.
1
Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan; 2Department of Immunology, Osaka City University Graduate School of Medicine, Osaka, Japan; 3Department of Dermatology, Yamagata University Faculty of Medicine, Yamagata, Japan; and 4Department of Dermatology, Osaka City General Hospital, Osaka, Japan Correspondence: Ayako Teramae, 1-4-3, Asahimachi, Abenoku, Osaka 5458585, Japan. E-mail: [email protected] Abbreviations: DOPA, 3,4-dihydroxy-L-phenylalanine; ER, endoplasmic reticulum; MBTH, 3-methyl-2-benzothiazoline hydrazine; OCA, oculocutaneous albinism; OCA1A, oculocutaneous albinism type 1A Received 27 June 2018; revised 24 September 2018; accepted 10 October 2018; accepted manuscript published online 14 November 2018; corrected proof published online XXX
Those sites are important for the catalytic activity of tyrosinase (Spritz et al., 1997). During its maturation process, tyrosinase is initially co-translationally translocated into the endoplasmic reticulum (ER) lumen. The nascent tyrosinase protein (w60 kDa) is then glycosylated and properly folded into a full-length protein (w70 kDa) and forms dimers (Francis et al., 2003), which are transported to the cis-Golgi apparatus for glycosylation. In the trans-Golgi network, the N-linked glycans are modified with further complex sugars, and the copper ions are bound (Wang and Hebert, 2006). The mature tyrosinase protein (w80 kDa) is then transferred from the trans-Golgi network to melanosomes. Mutations in TYR cause OCA, which is characterized by the absence or reduction of melanin synthesis in melanocytes by several mechanisms (King et al., 1991; Matsunaga et al., 1999; Tripathi et al., 1992). It was shown that missense mutant tyrosinase proteins are not properly folded in the ER and are not transported from the ER to the cis-Golgi apparatus and that their catalytic activity is partially or completely lost (Halaban et al., 2000; Toyofuku et al., 2001), a phenomenon known as ER retention. Chemical chaperone therapy can be effective for treating ER retention diseases, such as Gaucher disease (Sawkar et al., 2002) and Fabry disease (Fan et al., 1999). Chemical chaperones, usually at low concentrations of competitive inhibitors for the enzyme involved, bind to misfolded protein enzymes and form stable complexes. Those complexes can then be transported to the Golgi apparatus for further processing and can rescue the catalytic activities of mutant enzymes. Among competitive tyrosinase inhibitors, deoxyarbutin (i.e., 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol) is a glucoside derivative of hydroquinone that is more
ª 2018 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
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Table 1. Tyrosinase Mutations Identified in Japanese Patients with OCA1 Mutation Detected
n
%
P310insC
26
45.63
R77Q
10
17.55
R278X
6
10.53
P431L
2
3.51
H211Y
2
3.51
R239W
1
1.75
D383N
1
1.75
G97V
1
1.75
R299S
1
1.75
IVS2
4
7.02
S44N
1
1.75
F95L
1
1.75
E398G
1
1.75
OCA1A
OCA1B
Not classified
effective and less toxic than hydroquinone (Chawla et al., 2008). Thirteen mutations in TYR in Japanese OCA1 patients have been reported (Table 1) (Goto et al., 2004; Miyamura et al., 2005; Okamura et al., 2014). Among them, there are five missense mutations in the most severe type, OCA type 1A (OCA1A) (R77Q, H211Y, R239W, D383N, and P431L), in which a chemical chaperone effect might occur. The intracellular localization of tyrosinase in Japanese patients with OCA1A missense mutations has not been investigated so far, and there is no current effective treatment for OCA. The purpose of this study was to investigate the subcellular localization of tyrosinase in Japanese patients with OCA1A and examine the possibility of using deoxyarbutin as a chemical chaperone. This technique may offer a promising therapeutic modality for OCA1A. RESULTS Sensitivity of mutant tyrosinases to PNGaseF and EndoH
To confirm the production of tyrosinase mutant proteins via gene expression, we performed Western blotting. Wild-type tyrosinase was detected as an approximately 80-kDa protein composed of mature and immature species, and all mutant tyrosinases were detected around 70 kDa (Figure 1a). As we expected, endogenous tyrosinase was not detected in HeLa cells. To examine the intracellular localization of wild-type and missense mutant tyrosinases, we digested them with PNGaseF and with EndoH. PNGaseF removes almost all types of N-linked carbohydrates, and therefore all glycosylated proteins are sensitive to PNGaseF. In contrast, EndoH removes high mannose and hybrid types of N-linked carbohydrates, and therefore proteins transported to the trans-Golgi network are resistant to EndoH, whereas proteins retained within the ER or the cis-Golgi apparatus are sensitive to EndoH. Wildtype and all mutant tyrosinases examined were sensitive to PNGaseF (Figure 1b), but only the missense mutant tyrosinases were sensitive to EndoH (Figure 1c), whereas the 2
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wild-type enzyme was resistant. These results suggest that all mutant tyrosinases examined were retained in the ER or cis-Golgi apparatus. Intracellular localization of tyrosinase in Japanese patients with OCA1A
To characterize the intracellular localization of wild-type and missense mutant tyrosinases, we performed immunohistochemical staining (Figure 2). Tyrosinase was stained green, the ER and cis-Golgi apparatus were stained red, and nuclei were stained blue. Co-localization of signals was observed as yellow. The wild-type tyrosinase was seen as round spots that did not co-localize with the ER and also as a reticular pattern that co-localized in the cis-Golgi apparatus. The R77Q, R239W, D383N, and P431L mutant tyrosinases co-localized with the ER but did not co-localize with the cis-Golgi apparatus. The H211Y mutant tyrosinase co-localized with the ER and the cis-Golgi apparatus. Effect of deoxyarbutin as a chemical chaperone
To evaluate the chemical chaperone effect of deoxyarbutin, we cultured HeLa cells that stably expressed each mutant tyrosinase in the absence or presence of deoxyarbutin (at 5, 10, and 20 mmol/L), and assessed tyrosinase activity using the 3-methyl-2-benzothiazoline hydrazine (MBTH) assay (Winder and Harris, 1991), which measures the 3,4dihydroxy-L-phenylalanine (DOPA) oxidase activity of tyrosinase. We evaluated the effects of the chemical chaperone as the ratio of each mutant tyrosinase compared with the activity of the wild-type enzyme (Figure 3). It has been reported that treatment with 50 mmol/L deoxyarbutin resulted in a 50% inhibition of melanocyte tyrosinase activity. A significant increase was observed in the activity of the P431L missense mutant tyrosinase treated with 20 mmol/L deoxyarbutin, and increases in the activity of H211Y and R239W mutant tyrosinases were also evident, although not at a statistically significant level. No increase was noted with the R77Q and D383N missense mutant tyrosinases. To further ascertain the effects of deoxyarbutin, we carried out immunohistochemical staining. After HeLa cells stably expressing each mutant tyrosinase were cultured with 20 mmol/L deoxyarbutin for 5 days, we transferred the cells to four-chamber slides, and 1 day later, we performed immunohistochemical staining. The R77Q, H211Y, R239W, and D383N missense mutant tyrosinases co-localized with the ER (Figure 4, yellow merged images). The P431L missense mutant tyrosinase did not co-localize with the ER after treatment with 20 mmol/L deoxyarbutin. The R77Q, R239W, and D383N missense mutant tyrosinases did not co-localize with the cis-Golgi apparatus, but the P431L and H211Y missense mutant tyrosinases did co-localize with the cisGolgi apparatus. DISCUSSION The first purpose of this study was to examine the intracellular localization of missense mutant tyrosinases (R77Q, H211Y, R239W, D383N, and P431L) in Japanese OCA1A patients. The results of immunohistochemical staining indicate that wild-type tyrosinase was not retained in the ER and was transported to the cis-Golgi apparatus for further processing, whereas the R77Q, R239W, D383N, and P431L mutant
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Chemical Chaperone Therapy for OCA1A Figure 1. PNGaseF and EndoH sensitivity of wild-type and mutant tyrosinases. (a) Wild-type and all mutant tyrosinases were detected. (b) Wild-type and all mutant tyrosinases were sensitive to PNGaseF. (c) All mutant tyrosinases were sensitive to EndoH, but the wild-type tyrosinase was resistant. R, PNGaseFor EndoH- resistant form; S, PNGaseFor EndoH- sensitive form.
tyrosinases were retained in the ER. The H211Y missense mutant tyrosinase was retained in the ER and was only partially transported to the cis-Golgi apparatus. Sensitivity to PNGaseF digestion showed that wild-type and all mutant tyrosinases were glycosylated proteins. Sensitivity to EndoH digestion showed that all mutant tyrosinases were retained in the ER or the cis-Golgi apparatus and were not transported to the trans-Golgi network, whereas wild-type tyrosinase was transported to the trans-Golgi network. These findings suggest that the cause of the loss of melanin production in OCA1A patients with R77Q, R239W, D383N, or P431L missense mutant tyrosinase is ER retention, but the H211Y missense mutant tyrosinase is not retained in the ER. The H211Y substitution is located at the CuA copper binding site and disrupts one of the three histidine residues involved in copper binding. Copper binding is necessary for catalytic activity, and the binding of two copper ions is cooperative (Spritz et al., 1997). Thus, we speculate that a copper ion cannot bind to the CuA site of H211Y tyrosinase (or binds with a reduced affinity) and that the catalytic activity is decreased.
Second, we examined the possibility of chemical chaperone therapy for OCA1A patients with these Japanese tyrosinase missense mutations. Chemical chaperones, usually at low concentrations of competitive inhibitors, have been shown to bind misfolded proteins and stabilize them. Properly folded proteins are transported from the ER to the Golgi apparatus at neutral pH, and protein-chaperone complexes are safely transported to lysosomes, where they dissociate under the acidic condition (Suzuki et al., 2009), and then the catalytic activity is rescued. Quite a few competitive inhibitors of tyrosinase have been reported (Chang, 2009), and some of them have been safely used as skin-lightening agents for human skin. Among them, deoxyarbutin is more effective and less toxic than hydroquinone (Boissy et al., 2005; Chawla et al., 2008) In this study, we showed that deoxyarbutin increased the DOPA oxidase activity of HeLa cells that stably express the tyrosinase P431L missense mutant. No significant changes were observed with R77Q, H211Y, R239W, and D383N. Recent studies have shown that chemical chaperone therapy is effective in vivo, even when there is www.jidonline.org
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Figure 2. Intracellular localization of wild-type and Japanese OCA1A missense mutant tyrosinases observed by confocal microscopy. Anti-Gm130p antibody (a marker for the cis-Golgi apparatus) was labeled with rhodamine (red). Wild-type and H211Y mutant tyrosinase co-localize with the marker for the cis-Golgi apparatus, but the other mutant tyrosinases do not co-localize with the cis-Golgi apparatus. Anti-tyrosinase antibodies were labeled with Alexa Flour 647 (green), and anti-calnexin antibodies (a marker for the ER) were labeled with Alexa Fluor 594 (red). All five mutant tyrosinases co-localized with calnexin, whereas wild-type tyrosinase was located as round spots and did not co-localize with calnexin. Scale bars ¼ 10 mm. ER, endoplasmic reticulum; OCA1A, oculocutaneous albinism type 1A.
only a 20%e35% recovery of enzyme activity in vitro for Gaucher disease (Lei et al., 2007) and for GM1-gangliosidosis (Higaki et al., 2011; Matsuda et al., 2003). From the results of our experiments, it is possible to speculate that a fraction of the misfolded P431L tyrosinase mutant proteins were able to form the stable deoxyarbutin-mutP431L-tyrosinase complexes and were translocated to lysosomes, escaping degradation in ER; then, deoxyarbutin-mutP431L-tyrosinase complexes
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dissociated in the acidic condition of the lysosomes, and tyrosinase enzyme activities were recovered. Furthermore, it has been shown that melanosomes are acidic (Brilliant, 2001) as lysosomes. Therefore, we expected that the chemical chaperone effect could be obtained in the melanosomes. Unfortunately, we could not observe any EndoH-resistant form in the P431L tyrosinase mutant after deoxyarbutin treatment in our experimental system (data not shown).
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Chemical Chaperone Therapy for OCA1A
R77Q
H211Y
0.3
0.2
0.1
0.3
0.2
0.1
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
0.2
0.1
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
D383N
0.3
0.2
0.1
0 Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
P431L
0.4 ratio to wild-type tyrosinase
ratio to wild-type tyrosinase
0.4
0.3
0
0
0
R239W
0.4 ratio to wild-type tyrosinase
0.4 ratio to wild-type tyrosinase
ratio to wild-type tyrosinase
0.4
*
0.3
0.2
0.1
0
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
Figure 3. Tyrosinase activity after deoxyarbutin treatment. HeLa cells stably expressing each Japanese OCA1 missense mutant tyrosinase were cultured with deoxyarbutin at various concentrations as noted for 5 days. They were then collected and equalized for protein content and then measured using the MBTH assay. The P431L mutant tyrosinase treated with 20 mmol/L deoxyarbutin showed a significant increase in tyrosinase activity (P < 0.05, t test). Deoxyarbutin caused no significant increase in activity with the other mutant tyrosinases. The x-axis indicates the concentration of deoxyarbutin. The y-axis indicates the ratio of each mutant compared with the activity of wild-type tyrosinase. Experiments were performed in triplicate and were repeated three times. Each bar represents the mean standard deviation. M, mol/L; MBTH, 3,4-dihydroxy-l-phenylalanine; OCA1, oculocutaneous albinism type 1.
Therefore, we infer that the tyrosinase transported to lysosomes is biochemically undetectable under our conditions. OCA1A is characterized by a complete lack of melanogenesis not only in the skin but also in the retina and iris. Therefore, vision impairment also significantly affects the quality of life of patients with OCA1A. Although deoxyarbutin has been used in vivo as a skin-lightening agent, its safety for systemic administration has not been established. However, it has been reported that captopril, which is used as an antihypertensive agent, is a competitive tyrosinase inhibitor in vitro (Espin and Wichers, 2001) and that miconazole, which is used as a regional antifungal agent, is a tyrosinase inhibitor in vitro (Mun et al., 2004). We are considering whether those two drugs might also be candidates as chemical chaperones for systemic administration in patients with OCA1A. A further important point is that TYR, DHICA oxidase (TRP1) and DOPAchrome tautomerase (TRP2) show high degrees of homology in their amino acid sequences (Cassady and Sturm, 1994). Therefore, deoxyarbutin may inhibit TRP1 and/or TRP2. Further study is needed to investigate the possible use of systemic treatment with deoxyarbutin and to determine its possible effects on TRP1 and/or TRP2 using P431L model mice. The feasibility of using captopril and/or miconazole as chemical chaperones for systemic administration also needs to be assessed. In conclusion, our study showed that R77Q, R239W, D383N, and P431L missense mutant tyrosinases remain in the ER, and therefore their catalytic activities are lost.
However, the pathogenesis of the H211Y mutant tyrosinase is related to the loss of or reduced binding capacity of a copper ion to that mutant. Furthermore, deoxyarbutin can act as a chemical chaperone for the P431L tyrosinase mutant, offering a promising treatment modality for recovering tyrosinase activities of OCA1A patients with certain types of missense mutations of TYR. MATERIALS AND METHODS Cells, cultures, and transfections HeLa cells were obtained from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Innovation, Osaka, Japan) and were cultured in DMEM high glucose with L-glutamine and phenol red (WAKO Pure Chemical Industries, Tokyo, Japan) containing 10% fetal bovine serum (BioSera, Kansas City, MO) and 1% antibiotics-antimycotic 100 (Thermo Fisher Scientific, Waltham, MA). Cells were transfected using a lentivirus vector, as detailed in the next section.
Plasmids Human wild-type tyrosinase and Japanese OCA1A missense mutant tyrosinases (R77Q, H211Y, R239W, D383N, and P431L) were made using Quick Change II Site-Directed Mutagenesis Kits (Agilent Technologies, Santa Clara, CA). Sequences of all mutant tyrosinases were verified by DNA sequencing. Each tyrosinase cDNA was transferred into a lentivirus vector, pCSⅡ-EF-MCS-IRES2-Venus (a gift from H. Miyoshi, RIKEN, Tsukuba, Japan). The production and purification of lentivirus particles were performed as previously described (Miyoshi et al., 1997, 1998). www.jidonline.org
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Figure 4. Intracellular localization of Japanese OCA1A missense mutant tyrosinases after deoxyarbutin treatment. HeLa cells stably expressing each Japanese OCA1 missense mutant tyrosinase were cultured with deoxyarbutin at 20 mmol/L for 5 days, and then immunohistochemical staining was performed as described for Figure 2. All mutant tyrosinases except the P431L mutant co-localized with the ER. The R77Q, R239W, and D383N mutant tyrosinases did not co-localize with the cis-Golgi apparatus. The P431L mutant tyrosinase did not co-localize with the ER (bottom panel). Scale bars ¼ 10 mm. ER, endoplasmic reticulum; OCA1A, oculocutaneous albinism type 1A.
EndoH digestion analysis
Each supernatant containing 20 mg protein, obtained as described for the EndoH digestion, was reacted with 1 ml PNGaseF (Promega) according to the manufacturer’s instructions.
was added to lysis buffer to increase the total volume of the reaction to 12 ml. Each sample was then added to 2.5 ml SDS-PAGE sample buffer and heated at 95 C for 5 minutes. These samples were separated by SDS-PAGE and then electrophoretically transferred to membranes. Blots were incubated with the following primary antibodies: a rabbit polyclonal IgG antibody to tyrosinase (#sc15341, 1:1,000; Santa Cruz Biotechnology, Dallas, TX) and an antibody to b-actin (#sc4778, 1:2,000; Santa Cruz Biotechnology) as a loading control, and the following secondary antibodies: a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (#65-6120, 1:4,000; Invitrogen, Carlsbad, CA) and a horseradish peroxidase-conjugated goat antimouse IgG secondary antibody (#62-6520, 1:4,000; Invitrogen) for 2.5 hours. Immunoreactivity was detected using Chemi-Lumi One Super (Nacalai Tesque, Tokyo, Japan).
Western blotting
Immunohistochemical staining
Cells were solubilized in lysis buffer (50 mmol/L HEPES [i.e., 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.9, 420 mmol/L NaCl, 0.5% NP40 containing protease inhibitors) on ice. Each lysate was centrifuged for 10 minutes at 15,000 r.p.m. at 4 C, and the supernatants were collected. After protein concentrations were measured and equalized, supernatants containing 10 mg protein were reacted with 1 ml EndoH (Promega, Madison, WI) according to the manufacturer’s instructions.
PNGaseF digestion analysis
Each supernatant containing 10 mg (from the EndoH digestion) or 20 mg (from the PNGaseF digestion) of protein, obtained as described, 6
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Cells were plated in four-well Lab-Tek Chamber Slides (Nalge Nunc International, Rochester, NY) and incubated for 24 hours. On the
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Chemical Chaperone Therapy for OCA1A next day, cells were fixed with 3.7% formaldehyde for 15 minutes at room temperature. After washing in phosphate buffered saline three times, cells were permeabilized with 0.5% Triton X-100 (WAKO Pure Chemical Industries) for 5 minutes and washed again. Cells were then incubated in 5% skim milk for 1 hour to avoid nonspecific reactions and then with an anti-tyrosinase mouse monoclonal IgG antibody (#sc20035, 1:100; Santa Cruz Biotechnology) and a rabbit polyclonal anti-calnexin antibody (ADI-SPA-860, 1:100; Enzo Life Sciences, Farmingdale, NY) for 1 hour to detect tyrosinase and the ER, respectively. Similarly, to detect tyrosinase and the cis-Golgi apparatus, cells were incubated with the anti-tyrosinase mouse monoclonal IgG antibody and an anti-GM130 polyclonal antibody (#PM061, 1:500;MBL, Tokyo, Japan). After washing with TBS-T (WAKO Pure Chemical Industries) three times, they were incubated with Alexa Fluor 647-labeled donkey anti-mouse IgG (#A-21235, 1:300; Invitrogen), which recognizes the tyrosinase antibody, and Alexa Fluor 594-labeled donkey-anti-rabbit IgG (#R-37119, 1:300; Invitrogen), which recognizes the ER or cis-Golgi apparatus antibody, for 1 hour. They were then washed in TBS-T, and nuclei were stained with 40 ,6-diamidino-2-phenylindole (i.e., DAPI). All processing was done at room temperature. The stained cells were observed using a confocal microscope, FLUOVIEW FV10i (Olympus, Tokyo, Japan).
Deoxyarbutin treatment Deoxyarbutin was obtained from Carbosynth Limited (Compton, Berkshire, UK) and was dissolved in 100% ethanol at a concentration of 100 mmol/L. The solution was filtered and then added to cell cultures at final concentrations of 5, 10 and 20 mmol/L. The growth medium was replaced with fresh medium containing each concentration of deoxyarbutin every day. After incubation for 5 days, cells were recovered using lysis buffer, as described. Experiments were in triplicate and were repeated three times.
MBTH assay Tyrosinase activity of treated lysates was measured as DOPA oxidase activity using the MBTH assay. The reaction mixture (total volume 180 ml) containing 80 ml assay buffer (100 mmol/L phosphate buffer [pH 7.1], 4% dimethylformamide), 6 mmol/L MBTH (3-methyl-2benzothiazoline hydrazine), and 1 mmol/L L-DOPA. Treated cell lysates containing 20 mg protein each were added with the reaction mixture on ice and then incubated at 37 C for 1 hour. Absorbance was measured at 490 nm spectrophotometrically.
Espin JC, Wichers HJ. Effect of captopril on mushroom tyrosinase activity in vitro. Biochim Biophys Acta 2001;1544(1e2):289e300. Fan JQ, Ishii S, Asano N, Suzuki Y. Accelerated transport and maturation of lysosomal a-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat Med 1999;5:112e5. Francis E, Wang N, Parag H, Halaban R, Hebert DN. Tyrosinase maturation and oligomerization in the endoplasmic reticulum require a melanocytespecific factor. J Biol Chem 2003;278:25607e17. Goto M, Sato-Matsumura KC, Sawamura D, Yokota K, Nakamura H, Shimizu H. Tyrosinase gene analysis in Japanese patients with oculocutaneous albinism. J Dermatol Sci 2004;35:215e20. Halaban R, Svedine S, Cheng E, Smicun Y, Aron R, Hebert DN. Endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism. Proc Natl Acad Sci USA 2000;97:5889e94. Higaki K, Li L, Bahrudin U, Okuzawa S, Takamuram A, Yamamoto K, et al. Chemical chaperone therapy: chaperone effect on mutant enzyme and cellular pathophysiology in b-galactosidase deficiency. Hum Mutat 2011;32:843e52. King RA, Mentink MM, Oetting WS. Non-random distribution of missense mutations within the human tyrosinase gene in type I (tyrosinase-related) oculocutaneous albinism. Mol Biol Med 1991;8:19e29. Lei K, Ninomiya H, Suzuki M, Inoue T, Sawa M, Iida M, et al. Enzyme enhancement activity of N-octyl-b-valienamine on b-glucosidase mutants associated with Gaucher disease. Biochim Biophys Acta 2007;1772:587e96. Lerner AB, Fitzpatrick TB, Calkins E, Summerson WH. Mammalian tyrosinase; the relationship of copper to enzymatic activity. J Biol Chem 1950;187: 793e802. Matsuda J, Suzuki O, Oshima A, Yamamoto Y, Noguchi A, Takimoto K, et al. Chemical chaperone therapy for brain pathology in GM1-gangliosidosis. Proc Natl Acad Sci USA 2003;100:15912e7. Matsunaga J, Dakeishi-Hara M, Tanita M, Nindl M, Nagata Y, Nakamura E, et al. A splicing mutation of the tyrosinase gene causes yellow oculocutaneous albinism in a Japanese patient with a pigmented phenotype. Dermatology 1999;199:124e9. Miyamura Y, Verma IC, Saxena R, Hoshi M, Murase A, Nakamura E, et al. Five novel mutations in tyrosinase gene of Japanese and Indian patients with oculocutaneous albinism type I (OCA1). J Invest Dermatol 2005;125: 397e8. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol 1998;72:8150e7. Miyoshi H, Takahashi M, Gage FH, Verma IM. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA 1997;94:10319e23. Mun YJ, Lee SW, Jeong HW, Lee KG, Kim JH, Woo WH. Inhibition effect of miconazole on melanogenesis. Bio Pharm Bull 2004;27:806e9. Okamura K, Yoshizawa J, Abe Y, Hanaoka K, Higashi N, Togawa Y, et al. Oculocutaneous albinism (OCA) in Japanese patients: five novel mutations. J Dermatol Sci 2014;74:173e4.
ACKNOWLEDGMENTS
Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW. Chemical chaperones increase the cellular activity of N370S b-glucosidase: a therapeutic strategy for Gaucher disease. Proc Natl Acad Sci USA 2002;99: 15428e33.
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI#16K10170 to KF).
Spritz RA, Ho L, Furumura M, Hearing VJ Jr. Mutational analysis of copper binding by human tyrosinase. J Invest Dermatol 1997;109:207e12.
CONFLICT OF INTEREST The authors state no conflict of interest.
REFERENCES Boissy RE, Visscher M, DeLong MA. DeoxyArbutin: a novel reversible tyrosinase inhibitor with effective in vivo skin lightening potency. Exp Dermatol 2005;14:601e8. Brilliant MH. The mouse p (pink-eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH. Pigment Cell Res 2001;14:86e93. Cassady JL, Sturm RA. Sequence of the human dopachrome tautomeraseencoding TRP-2 cDNA. Gene 1994;143:295e8. Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci 2009;10: 2440e75. Chawla S, deLong MA, Visscher MO, Wickett RR, Manga P, Boissy RE. Mechanism of tyrosinase inhibition by deoxyArbutin and its secondgeneration derivatives. Br J Dermatol 2008;159:1267e74.
Suzuki Y, Ogawa S, Sakakibara Y. Chaperone therapy for neuronopathic lysosomal diseases: competitive inhibitors as chemical chaperones for enhancement of mutant enzyme activities. Perspect Medicin Chem 2009;3: 7e19. Toyofuku K, Wada I, Valencia JC, Kushimoto T, Ferrans VJ, Hearing VJ. Oculocutaneous albinism types 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wildtype proteins. FASEB J 2001;15:2149e61. Tripathi RK, Strunk KM, Giebel LB, Weleber RG, Spritz RA. Tyrosinase gene mutations in type I (tyrosinase-deficient) oculocutaneous albinism define two clusters of missense substitutions. Am J Med Genet 1992;43:865e71. Wang N, Hebert DN. Tyrosinase maturation through the mammalian secretory pathway: bringing color to life. Pigment Cell Res 2006;19:3e18. Winder AJ, Harris H. New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. Eur J Biochem 1991;198:317e26.
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The Molecular Basis of Chemical Chaperone Therapy for Oculocutaneous Albinism Type 1A Ayako Teramae1, Yui Kobayashi1, Hiroyuki Kunimoto2, Koichi Nakajima2, Tamio Suzuki3, Daisuke Tsuruta1 and Kazuyoshi Fukai4 Oculocutaneous albinism (OCA) is an autosomal recessive disease characterized by the reduction or complete lack of melanin pigment in the skin, hair, and eyes. No effective treatment for OCA is available at present. OCA type 1 is caused by mutations that disrupt the function of tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis. Recently, it was shown that tyrosinase in some patients with OCA type 1 mutation is retained in the endoplasmic reticulum and that its catalytic activity is lost, a phenomenon known as endoplasmic reticulum retention. However, to our knowledge, the intracellular localization of tyrosinase in Japanese patients with OCA type 1 missense mutations has not been reported. In this study, we first investigated the intracellular localization of Japanese OCA type 1A missense mutant tyrosinases using Western blotting and immunohistochemical staining. R77Q, R239W, D383N, and P431L mutant tyrosinases were retained in the endoplasmic reticulum, and H211Y mutant tyrosinase was partially transported to the Golgi apparatus. Second, we explored the possibility of chemical chaperone therapy for Japanese patients with OCA type 1A missense mutations and found that HeLa cells expressing P431L mutant tyrosinase have restored tyrosinase activity after treatment with a low-dose tyrosinase inhibitor, as a chemical chaperone, in a dose-dependent manner. These results provide the basis for a possible chemical chaperone therapy to recover tyrosinase activities in patients with OCA type 1A patients. Journal of Investigative Dermatology (2018) -, -e-; doi:10.1016/j.jid.2018.10.033
INTRODUCTION Oculocutaneous albinism (OCA) is a group of autosomal recessive diseases of pigmentation caused by the complete lack or reduction of melanin biosynthesis in melanocytes. Seven types of OCA have been classified according to the genes responsible. Among them, OCA type 1 (OCA1) is characterized by hypopigmentation of the skin, hair, and eyes. OCA1 is caused by mutations in the tyrosinase gene (TYR). Tyrosinase is a copper-containing enzyme protein that is critically involved in the biosynthesis of melanin. Human tyrosinase is a type 1 membrane glycoprotein that contains 529 amino acids with a N-terminal signal sequence and a C-terminal transmembrane domain. It has seven asparagine-linked (N-linked) glycosylation sites (Wang and Hebert, 2006) and two copper binding sites, CuA and CuB. Those two sites each contain three histidine residues that coordinate the binding of copper (Lerner et al., 1950), CuA at His 180, 202, and 211 and CuB at His 363, 367, and 389.
1
Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan; 2Department of Immunology, Osaka City University Graduate School of Medicine, Osaka, Japan; 3Department of Dermatology, Yamagata University Faculty of Medicine, Yamagata, Japan; and 4Department of Dermatology, Osaka City General Hospital, Osaka, Japan Correspondence: Ayako Teramae, 1-4-3, Asahimachi, Abenoku, Osaka 5458585, Japan. E-mail: [email protected] Abbreviations: DOPA, 3,4-dihydroxy-L-phenylalanine; ER, endoplasmic reticulum; MBTH, 3-methyl-2-benzothiazoline hydrazine; OCA, oculocutaneous albinism; OCA1A, oculocutaneous albinism type 1A Received 27 June 2018; revised 24 September 2018; accepted 10 October 2018; accepted manuscript published online 14 November 2018; corrected proof published online XXX
Those sites are important for the catalytic activity of tyrosinase (Spritz et al., 1997). During its maturation process, tyrosinase is initially co-translationally translocated into the endoplasmic reticulum (ER) lumen. The nascent tyrosinase protein (w60 kDa) is then glycosylated and properly folded into a full-length protein (w70 kDa) and forms dimers (Francis et al., 2003), which are transported to the cis-Golgi apparatus for glycosylation. In the trans-Golgi network, the N-linked glycans are modified with further complex sugars, and the copper ions are bound (Wang and Hebert, 2006). The mature tyrosinase protein (w80 kDa) is then transferred from the trans-Golgi network to melanosomes. Mutations in TYR cause OCA, which is characterized by the absence or reduction of melanin synthesis in melanocytes by several mechanisms (King et al., 1991; Matsunaga et al., 1999; Tripathi et al., 1992). It was shown that missense mutant tyrosinase proteins are not properly folded in the ER and are not transported from the ER to the cis-Golgi apparatus and that their catalytic activity is partially or completely lost (Halaban et al., 2000; Toyofuku et al., 2001), a phenomenon known as ER retention. Chemical chaperone therapy can be effective for treating ER retention diseases, such as Gaucher disease (Sawkar et al., 2002) and Fabry disease (Fan et al., 1999). Chemical chaperones, usually at low concentrations of competitive inhibitors for the enzyme involved, bind to misfolded protein enzymes and form stable complexes. Those complexes can then be transported to the Golgi apparatus for further processing and can rescue the catalytic activities of mutant enzymes. Among competitive tyrosinase inhibitors, deoxyarbutin (i.e., 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol) is a glucoside derivative of hydroquinone that is more
ª 2018 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
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Table 1. Tyrosinase Mutations Identified in Japanese Patients with OCA1 Mutation Detected
n
%
P310insC
26
45.63
R77Q
10
17.55
R278X
6
10.53
P431L
2
3.51
H211Y
2
3.51
R239W
1
1.75
D383N
1
1.75
G97V
1
1.75
R299S
1
1.75
IVS2
4
7.02
S44N
1
1.75
F95L
1
1.75
E398G
1
1.75
OCA1A
OCA1B
Not classified
effective and less toxic than hydroquinone (Chawla et al., 2008). Thirteen mutations in TYR in Japanese OCA1 patients have been reported (Table 1) (Goto et al., 2004; Miyamura et al., 2005; Okamura et al., 2014). Among them, there are five missense mutations in the most severe type, OCA type 1A (OCA1A) (R77Q, H211Y, R239W, D383N, and P431L), in which a chemical chaperone effect might occur. The intracellular localization of tyrosinase in Japanese patients with OCA1A missense mutations has not been investigated so far, and there is no current effective treatment for OCA. The purpose of this study was to investigate the subcellular localization of tyrosinase in Japanese patients with OCA1A and examine the possibility of using deoxyarbutin as a chemical chaperone. This technique may offer a promising therapeutic modality for OCA1A. RESULTS Sensitivity of mutant tyrosinases to PNGaseF and EndoH
To confirm the production of tyrosinase mutant proteins via gene expression, we performed Western blotting. Wild-type tyrosinase was detected as an approximately 80-kDa protein composed of mature and immature species, and all mutant tyrosinases were detected around 70 kDa (Figure 1a). As we expected, endogenous tyrosinase was not detected in HeLa cells. To examine the intracellular localization of wild-type and missense mutant tyrosinases, we digested them with PNGaseF and with EndoH. PNGaseF removes almost all types of N-linked carbohydrates, and therefore all glycosylated proteins are sensitive to PNGaseF. In contrast, EndoH removes high mannose and hybrid types of N-linked carbohydrates, and therefore proteins transported to the trans-Golgi network are resistant to EndoH, whereas proteins retained within the ER or the cis-Golgi apparatus are sensitive to EndoH. Wildtype and all mutant tyrosinases examined were sensitive to PNGaseF (Figure 1b), but only the missense mutant tyrosinases were sensitive to EndoH (Figure 1c), whereas the 2
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wild-type enzyme was resistant. These results suggest that all mutant tyrosinases examined were retained in the ER or cis-Golgi apparatus. Intracellular localization of tyrosinase in Japanese patients with OCA1A
To characterize the intracellular localization of wild-type and missense mutant tyrosinases, we performed immunohistochemical staining (Figure 2). Tyrosinase was stained green, the ER and cis-Golgi apparatus were stained red, and nuclei were stained blue. Co-localization of signals was observed as yellow. The wild-type tyrosinase was seen as round spots that did not co-localize with the ER and also as a reticular pattern that co-localized in the cis-Golgi apparatus. The R77Q, R239W, D383N, and P431L mutant tyrosinases co-localized with the ER but did not co-localize with the cis-Golgi apparatus. The H211Y mutant tyrosinase co-localized with the ER and the cis-Golgi apparatus. Effect of deoxyarbutin as a chemical chaperone
To evaluate the chemical chaperone effect of deoxyarbutin, we cultured HeLa cells that stably expressed each mutant tyrosinase in the absence or presence of deoxyarbutin (at 5, 10, and 20 mmol/L), and assessed tyrosinase activity using the 3-methyl-2-benzothiazoline hydrazine (MBTH) assay (Winder and Harris, 1991), which measures the 3,4dihydroxy-L-phenylalanine (DOPA) oxidase activity of tyrosinase. We evaluated the effects of the chemical chaperone as the ratio of each mutant tyrosinase compared with the activity of the wild-type enzyme (Figure 3). It has been reported that treatment with 50 mmol/L deoxyarbutin resulted in a 50% inhibition of melanocyte tyrosinase activity. A significant increase was observed in the activity of the P431L missense mutant tyrosinase treated with 20 mmol/L deoxyarbutin, and increases in the activity of H211Y and R239W mutant tyrosinases were also evident, although not at a statistically significant level. No increase was noted with the R77Q and D383N missense mutant tyrosinases. To further ascertain the effects of deoxyarbutin, we carried out immunohistochemical staining. After HeLa cells stably expressing each mutant tyrosinase were cultured with 20 mmol/L deoxyarbutin for 5 days, we transferred the cells to four-chamber slides, and 1 day later, we performed immunohistochemical staining. The R77Q, H211Y, R239W, and D383N missense mutant tyrosinases co-localized with the ER (Figure 4, yellow merged images). The P431L missense mutant tyrosinase did not co-localize with the ER after treatment with 20 mmol/L deoxyarbutin. The R77Q, R239W, and D383N missense mutant tyrosinases did not co-localize with the cis-Golgi apparatus, but the P431L and H211Y missense mutant tyrosinases did co-localize with the cisGolgi apparatus. DISCUSSION The first purpose of this study was to examine the intracellular localization of missense mutant tyrosinases (R77Q, H211Y, R239W, D383N, and P431L) in Japanese OCA1A patients. The results of immunohistochemical staining indicate that wild-type tyrosinase was not retained in the ER and was transported to the cis-Golgi apparatus for further processing, whereas the R77Q, R239W, D383N, and P431L mutant
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Chemical Chaperone Therapy for OCA1A Figure 1. PNGaseF and EndoH sensitivity of wild-type and mutant tyrosinases. (a) Wild-type and all mutant tyrosinases were detected. (b) Wild-type and all mutant tyrosinases were sensitive to PNGaseF. (c) All mutant tyrosinases were sensitive to EndoH, but the wild-type tyrosinase was resistant. R, PNGaseFor EndoH- resistant form; S, PNGaseFor EndoH- sensitive form.
tyrosinases were retained in the ER. The H211Y missense mutant tyrosinase was retained in the ER and was only partially transported to the cis-Golgi apparatus. Sensitivity to PNGaseF digestion showed that wild-type and all mutant tyrosinases were glycosylated proteins. Sensitivity to EndoH digestion showed that all mutant tyrosinases were retained in the ER or the cis-Golgi apparatus and were not transported to the trans-Golgi network, whereas wild-type tyrosinase was transported to the trans-Golgi network. These findings suggest that the cause of the loss of melanin production in OCA1A patients with R77Q, R239W, D383N, or P431L missense mutant tyrosinase is ER retention, but the H211Y missense mutant tyrosinase is not retained in the ER. The H211Y substitution is located at the CuA copper binding site and disrupts one of the three histidine residues involved in copper binding. Copper binding is necessary for catalytic activity, and the binding of two copper ions is cooperative (Spritz et al., 1997). Thus, we speculate that a copper ion cannot bind to the CuA site of H211Y tyrosinase (or binds with a reduced affinity) and that the catalytic activity is decreased.
Second, we examined the possibility of chemical chaperone therapy for OCA1A patients with these Japanese tyrosinase missense mutations. Chemical chaperones, usually at low concentrations of competitive inhibitors, have been shown to bind misfolded proteins and stabilize them. Properly folded proteins are transported from the ER to the Golgi apparatus at neutral pH, and protein-chaperone complexes are safely transported to lysosomes, where they dissociate under the acidic condition (Suzuki et al., 2009), and then the catalytic activity is rescued. Quite a few competitive inhibitors of tyrosinase have been reported (Chang, 2009), and some of them have been safely used as skin-lightening agents for human skin. Among them, deoxyarbutin is more effective and less toxic than hydroquinone (Boissy et al., 2005; Chawla et al., 2008) In this study, we showed that deoxyarbutin increased the DOPA oxidase activity of HeLa cells that stably express the tyrosinase P431L missense mutant. No significant changes were observed with R77Q, H211Y, R239W, and D383N. Recent studies have shown that chemical chaperone therapy is effective in vivo, even when there is www.jidonline.org
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Figure 2. Intracellular localization of wild-type and Japanese OCA1A missense mutant tyrosinases observed by confocal microscopy. Anti-Gm130p antibody (a marker for the cis-Golgi apparatus) was labeled with rhodamine (red). Wild-type and H211Y mutant tyrosinase co-localize with the marker for the cis-Golgi apparatus, but the other mutant tyrosinases do not co-localize with the cis-Golgi apparatus. Anti-tyrosinase antibodies were labeled with Alexa Flour 647 (green), and anti-calnexin antibodies (a marker for the ER) were labeled with Alexa Fluor 594 (red). All five mutant tyrosinases co-localized with calnexin, whereas wild-type tyrosinase was located as round spots and did not co-localize with calnexin. Scale bars ¼ 10 mm. ER, endoplasmic reticulum; OCA1A, oculocutaneous albinism type 1A.
only a 20%e35% recovery of enzyme activity in vitro for Gaucher disease (Lei et al., 2007) and for GM1-gangliosidosis (Higaki et al., 2011; Matsuda et al., 2003). From the results of our experiments, it is possible to speculate that a fraction of the misfolded P431L tyrosinase mutant proteins were able to form the stable deoxyarbutin-mutP431L-tyrosinase complexes and were translocated to lysosomes, escaping degradation in ER; then, deoxyarbutin-mutP431L-tyrosinase complexes
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dissociated in the acidic condition of the lysosomes, and tyrosinase enzyme activities were recovered. Furthermore, it has been shown that melanosomes are acidic (Brilliant, 2001) as lysosomes. Therefore, we expected that the chemical chaperone effect could be obtained in the melanosomes. Unfortunately, we could not observe any EndoH-resistant form in the P431L tyrosinase mutant after deoxyarbutin treatment in our experimental system (data not shown).
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R77Q
H211Y
0.3
0.2
0.1
0.3
0.2
0.1
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
0.2
0.1
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
D383N
0.3
0.2
0.1
0 Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
P431L
0.4 ratio to wild-type tyrosinase
ratio to wild-type tyrosinase
0.4
0.3
0
0
0
R239W
0.4 ratio to wild-type tyrosinase
0.4 ratio to wild-type tyrosinase
ratio to wild-type tyrosinase
0.4
*
0.3
0.2
0.1
0
Ethanol 5 µmol/L 10 µmol/L 20 µmol/L
Figure 3. Tyrosinase activity after deoxyarbutin treatment. HeLa cells stably expressing each Japanese OCA1 missense mutant tyrosinase were cultured with deoxyarbutin at various concentrations as noted for 5 days. They were then collected and equalized for protein content and then measured using the MBTH assay. The P431L mutant tyrosinase treated with 20 mmol/L deoxyarbutin showed a significant increase in tyrosinase activity (P < 0.05, t test). Deoxyarbutin caused no significant increase in activity with the other mutant tyrosinases. The x-axis indicates the concentration of deoxyarbutin. The y-axis indicates the ratio of each mutant compared with the activity of wild-type tyrosinase. Experiments were performed in triplicate and were repeated three times. Each bar represents the mean standard deviation. M, mol/L; MBTH, 3,4-dihydroxy-l-phenylalanine; OCA1, oculocutaneous albinism type 1.
Therefore, we infer that the tyrosinase transported to lysosomes is biochemically undetectable under our conditions. OCA1A is characterized by a complete lack of melanogenesis not only in the skin but also in the retina and iris. Therefore, vision impairment also significantly affects the quality of life of patients with OCA1A. Although deoxyarbutin has been used in vivo as a skin-lightening agent, its safety for systemic administration has not been established. However, it has been reported that captopril, which is used as an antihypertensive agent, is a competitive tyrosinase inhibitor in vitro (Espin and Wichers, 2001) and that miconazole, which is used as a regional antifungal agent, is a tyrosinase inhibitor in vitro (Mun et al., 2004). We are considering whether those two drugs might also be candidates as chemical chaperones for systemic administration in patients with OCA1A. A further important point is that TYR, DHICA oxidase (TRP1) and DOPAchrome tautomerase (TRP2) show high degrees of homology in their amino acid sequences (Cassady and Sturm, 1994). Therefore, deoxyarbutin may inhibit TRP1 and/or TRP2. Further study is needed to investigate the possible use of systemic treatment with deoxyarbutin and to determine its possible effects on TRP1 and/or TRP2 using P431L model mice. The feasibility of using captopril and/or miconazole as chemical chaperones for systemic administration also needs to be assessed. In conclusion, our study showed that R77Q, R239W, D383N, and P431L missense mutant tyrosinases remain in the ER, and therefore their catalytic activities are lost.
However, the pathogenesis of the H211Y mutant tyrosinase is related to the loss of or reduced binding capacity of a copper ion to that mutant. Furthermore, deoxyarbutin can act as a chemical chaperone for the P431L tyrosinase mutant, offering a promising treatment modality for recovering tyrosinase activities of OCA1A patients with certain types of missense mutations of TYR. MATERIALS AND METHODS Cells, cultures, and transfections HeLa cells were obtained from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Innovation, Osaka, Japan) and were cultured in DMEM high glucose with L-glutamine and phenol red (WAKO Pure Chemical Industries, Tokyo, Japan) containing 10% fetal bovine serum (BioSera, Kansas City, MO) and 1% antibiotics-antimycotic 100 (Thermo Fisher Scientific, Waltham, MA). Cells were transfected using a lentivirus vector, as detailed in the next section.
Plasmids Human wild-type tyrosinase and Japanese OCA1A missense mutant tyrosinases (R77Q, H211Y, R239W, D383N, and P431L) were made using Quick Change II Site-Directed Mutagenesis Kits (Agilent Technologies, Santa Clara, CA). Sequences of all mutant tyrosinases were verified by DNA sequencing. Each tyrosinase cDNA was transferred into a lentivirus vector, pCSⅡ-EF-MCS-IRES2-Venus (a gift from H. Miyoshi, RIKEN, Tsukuba, Japan). The production and purification of lentivirus particles were performed as previously described (Miyoshi et al., 1997, 1998). www.jidonline.org
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Figure 4. Intracellular localization of Japanese OCA1A missense mutant tyrosinases after deoxyarbutin treatment. HeLa cells stably expressing each Japanese OCA1 missense mutant tyrosinase were cultured with deoxyarbutin at 20 mmol/L for 5 days, and then immunohistochemical staining was performed as described for Figure 2. All mutant tyrosinases except the P431L mutant co-localized with the ER. The R77Q, R239W, and D383N mutant tyrosinases did not co-localize with the cis-Golgi apparatus. The P431L mutant tyrosinase did not co-localize with the ER (bottom panel). Scale bars ¼ 10 mm. ER, endoplasmic reticulum; OCA1A, oculocutaneous albinism type 1A.
EndoH digestion analysis
Each supernatant containing 20 mg protein, obtained as described for the EndoH digestion, was reacted with 1 ml PNGaseF (Promega) according to the manufacturer’s instructions.
was added to lysis buffer to increase the total volume of the reaction to 12 ml. Each sample was then added to 2.5 ml SDS-PAGE sample buffer and heated at 95 C for 5 minutes. These samples were separated by SDS-PAGE and then electrophoretically transferred to membranes. Blots were incubated with the following primary antibodies: a rabbit polyclonal IgG antibody to tyrosinase (#sc15341, 1:1,000; Santa Cruz Biotechnology, Dallas, TX) and an antibody to b-actin (#sc4778, 1:2,000; Santa Cruz Biotechnology) as a loading control, and the following secondary antibodies: a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (#65-6120, 1:4,000; Invitrogen, Carlsbad, CA) and a horseradish peroxidase-conjugated goat antimouse IgG secondary antibody (#62-6520, 1:4,000; Invitrogen) for 2.5 hours. Immunoreactivity was detected using Chemi-Lumi One Super (Nacalai Tesque, Tokyo, Japan).
Western blotting
Immunohistochemical staining
Cells were solubilized in lysis buffer (50 mmol/L HEPES [i.e., 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.9, 420 mmol/L NaCl, 0.5% NP40 containing protease inhibitors) on ice. Each lysate was centrifuged for 10 minutes at 15,000 r.p.m. at 4 C, and the supernatants were collected. After protein concentrations were measured and equalized, supernatants containing 10 mg protein were reacted with 1 ml EndoH (Promega, Madison, WI) according to the manufacturer’s instructions.
PNGaseF digestion analysis
Each supernatant containing 10 mg (from the EndoH digestion) or 20 mg (from the PNGaseF digestion) of protein, obtained as described, 6
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Cells were plated in four-well Lab-Tek Chamber Slides (Nalge Nunc International, Rochester, NY) and incubated for 24 hours. On the
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Chemical Chaperone Therapy for OCA1A next day, cells were fixed with 3.7% formaldehyde for 15 minutes at room temperature. After washing in phosphate buffered saline three times, cells were permeabilized with 0.5% Triton X-100 (WAKO Pure Chemical Industries) for 5 minutes and washed again. Cells were then incubated in 5% skim milk for 1 hour to avoid nonspecific reactions and then with an anti-tyrosinase mouse monoclonal IgG antibody (#sc20035, 1:100; Santa Cruz Biotechnology) and a rabbit polyclonal anti-calnexin antibody (ADI-SPA-860, 1:100; Enzo Life Sciences, Farmingdale, NY) for 1 hour to detect tyrosinase and the ER, respectively. Similarly, to detect tyrosinase and the cis-Golgi apparatus, cells were incubated with the anti-tyrosinase mouse monoclonal IgG antibody and an anti-GM130 polyclonal antibody (#PM061, 1:500;MBL, Tokyo, Japan). After washing with TBS-T (WAKO Pure Chemical Industries) three times, they were incubated with Alexa Fluor 647-labeled donkey anti-mouse IgG (#A-21235, 1:300; Invitrogen), which recognizes the tyrosinase antibody, and Alexa Fluor 594-labeled donkey-anti-rabbit IgG (#R-37119, 1:300; Invitrogen), which recognizes the ER or cis-Golgi apparatus antibody, for 1 hour. They were then washed in TBS-T, and nuclei were stained with 40 ,6-diamidino-2-phenylindole (i.e., DAPI). All processing was done at room temperature. The stained cells were observed using a confocal microscope, FLUOVIEW FV10i (Olympus, Tokyo, Japan).
Deoxyarbutin treatment Deoxyarbutin was obtained from Carbosynth Limited (Compton, Berkshire, UK) and was dissolved in 100% ethanol at a concentration of 100 mmol/L. The solution was filtered and then added to cell cultures at final concentrations of 5, 10 and 20 mmol/L. The growth medium was replaced with fresh medium containing each concentration of deoxyarbutin every day. After incubation for 5 days, cells were recovered using lysis buffer, as described. Experiments were in triplicate and were repeated three times.
MBTH assay Tyrosinase activity of treated lysates was measured as DOPA oxidase activity using the MBTH assay. The reaction mixture (total volume 180 ml) containing 80 ml assay buffer (100 mmol/L phosphate buffer [pH 7.1], 4% dimethylformamide), 6 mmol/L MBTH (3-methyl-2benzothiazoline hydrazine), and 1 mmol/L L-DOPA. Treated cell lysates containing 20 mg protein each were added with the reaction mixture on ice and then incubated at 37 C for 1 hour. Absorbance was measured at 490 nm spectrophotometrically.
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ACKNOWLEDGMENTS
Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW. Chemical chaperones increase the cellular activity of N370S b-glucosidase: a therapeutic strategy for Gaucher disease. Proc Natl Acad Sci USA 2002;99: 15428e33.
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI#16K10170 to KF).
Spritz RA, Ho L, Furumura M, Hearing VJ Jr. Mutational analysis of copper binding by human tyrosinase. J Invest Dermatol 1997;109:207e12.
CONFLICT OF INTEREST The authors state no conflict of interest.
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