Original Research Article

Correction of Defective Early Tyrosinase Processing by Bafilomycin A1

and Monensin in Pink-Eyed Dilution Melanocytes

KUN CHEN, LJILJANA MINWALLA, LI NI and SETH J. ORLOW

The Ronald O. Perelman Department of Dermatology and The Department of Cell Biology, New York University School of Medicine, New York,

NY, USA*Address reprint requests to Seth J. Orlow, Dermatology Room H-100, NYU School of Medicine, 560 First Avenue, New York, NY 10016, USA.E-mail: seth.orlow@med.nyu.edu

Received 8 August 2003; in final form 23 September 2003

Mutations in the human P gene result in oculocutaneous

albinism type 2, the most common form of albinism. Mouse

melan-p1 melanocytes, cultured from mice null at the homol-

ogous pink-eyed dilution (p) locus, exhibit defective melanin

production. A variety of compounds including tyrosine, NH4Cl,

bafilomycin A1, concanamycin, monensin, and nigericin are

capable of restoring melanin synthesis in these cells. In the

current study, we investigated the subcellular effects of

bafilomycin A1 and monensin treatment of melan-p1 cells.

Both agents play two roles in the processing of tyrosinase (Tyr)

in melan-p1 cells. First, combined glycosidase digestion and

immunoblotting analysis showed that these agents reduce levels

of Tyr retained in the endoplasmic reticulum (ER) and

facilitate the release of Tyr from the ER to the Golgi.

Secondly, treatment with these compounds resulted in the

stabilization of Tyr. Surprisingly, induction of melanin syn-

thesis corresponds more closely with diminution of ER-retained

Tyr, rather than the absolute amount of Tyr. Our results

suggest that bafilomycin A1 and monensin induce melanin

synthesis in melan-p1 cells mainly by facilitating Tyr process-

ing from the ER to the Golgi by increasing the pH in either the

ER or the ER–Golgi intermediate compartment.

Key words: Bafilomycin A1, Monensin, Tyrosinase, OCA2,

Melanosomes

INTRODUCTION

The most common form of albinism in the world isoculocutaneous albinism type 2 (OCA2), caused by muta-tions in the P gene (1). Individuals with OCA2 exhibithypopigmentation of the skin, hair, and eyes. Affected

persons also demonstrate nystagmus and reduced visualacuity. Mutations in the homologous murine pink-eyeddilution (p) gene causes a similar phenotype in mice (2).

Recently, it has been shown that the p gene product controlsthe early processing of tyrosinase (Tyr) (3, 4). Tyr plays a keyrole in melanin synthesis, catalyzing the conversion of tyrosine

to dopaquinone. Tyr is a type I membrane protein with a largelumenal enzymatic domain and a small cytoplasmic domaincontaining signals necessary for appropriate trafficking (5).

Mutations in the Tyr gene are associated with oculocutaneousalbinism type I. Multiple Tyr defects are observed in mouse

melanocytes (melan-p1 cells), cultured from p-null mice(3, 4, 6). A high percentage of Tyr is trapped in theendoplasmic reticulum (ER) and Tyr has a shorter half-lifein melan-p1 cells. This accelerated turnover rate is specific to

Tyr in melan-p1 cells because the half-lives of other melano-somal proteins do not differ between mouse melanocyte wild-type at p locus (melan-a) and melan-p1 cells (4). Tyr in melan-

p1 cells is localized to a perinuclear compartment rather thanto melanosomes (3, 6, 7). Mislocalized Tyr is subsequentlycleaved and secreted into the culture medium. A truncated

Tyr, devoid of its carboxy terminus, can be detected in theculture medium of melan-p1 cells, but not in that of melan-acells (4). These defects can be corrected by transfection of

melan-p1 cells with an epitope-tagged wild-type p construct,indicating that the defects observed are the direct result of the

Abbreviations – ER, endoplasmic reticulum; Melan-a, mouse melanocyte wild-type at p locus; melan-p1, mouse melanocyte with p mutation; OCA2,oculocutaneous albinism type 2; p, mouse pink-eyed dilution protein; Tyr, mouse tyrosinase; Tyrp1, mouse tyrosinase related protein 1; V-ATPase,vacuolar-type (H+)-ATPase

PIGMENT CELL RES 17: 36–42. 2004 Copyright � Blackwell Munksgaard 2004

Printed in UK—all rights reserved

36 Pigment Cell Res. 17, 2004

loss of p function (3, 8). In addition, we found that themajority of p protein is located in the ER, supporting afunctional role for p within the ER compartment (3).

It has been shown that four distinct groups of compoundsare able to induce melanin formation in melan-p1 cells (3, 8,9, 10). High concentrations of tyrosine, the substrate for Tyr,are capable of stimulating melanin synthesis in melan-p1 cells

(7, 10, 11). Vacuolar-type (H+)-ATPase (V-ATPase) inhib-itors bafilomycin A1 and concanamycin A are potentstimulators, effective at nanomolar concentrations (3, 8, 9).

V-ATPases are known to play a role in pumping H+ ionsinto various endomembrane systems and cellular organellessuch as endosomes and lysosomes (12). Ionophores such as

monensin and nigericin can induce melanin synthesis inmelan-p1 cells at micromolar concentrations (9). Monensinand nigericin are best known to disrupt Na+/H+ exchange

and K+/H+ exchange in the Golgi, respectively (13). NH4Clis also capable of melanin induction in melan-p1 cells (3, 9).

The means by which such a diverse group of compounds canstimulate melanin synthesis in melan-p1 cells is not known.

While NH4Cl, V-ATPase inhibitors and ionophores can affectthe pH of various subcellular compartments, tyrosine has notbeen shown to do so. Ancans et al. (9) proposed that

melanosomes of melan-p1 cells were too acidic to allowmelanin synthesis. Addition of the above compounds washypothesized to neutralize the pHofmelanosomes and thereby

allow melanin synthesis to proceed (9, 14). However, there isno direct evidence that the site of action of these compounds isat the level of the melanosome, and defective Tyr processing inmelan-p1 cells is seen as early as the ER compartment.

In the current study, we explored how bafilomycin A1 andmonensin induce melanin synthesis by analyzing their effectsupon Tyr processing in melan-p1 cells. We find that both

agents modify Tyr processing and correct Tyr trafficking tomelanosomes. The ability to induce melanin synthesiscorresponds best with each agent’s ability to reverse the

aberrant ER retention of Tyr observed in melan-p1 cells. Themelanogenic effects of both compounds in melan-p1 cellsare consistent with a modification of the ER or ER–Golgi

intermediate compartment (ERGIC).

MATERIALS AND METHODS

Cell Culture

Melan-a (a/a,P/P) is an immortalized melanocyte line derived

from black C57BL/6J mice wild-type at the p locus (15).Melan-p1 is an immortalized melanocyte line from �OCA2�mice lacking p gene transcripts as a result of overlapping

deletions (11) (a/a, pcp/p25H). Cells were maintained in RPMImedium (Sigma, St Louis, MO, USA) as previously described(8). Where indicated, bafilomycin A1 (Wako, Richmond, VA,

USA), monensin or 3-isobutyl-1-methylxanthine (IBMX)(Sigma) was added to cell cultures for 48 h.

Antibodies

aPep7 antiserum was raised against the c-terminus of mouse

Tyr (Genemed, San Francisco, CA, USA) based on pub-lished antigenic peptide sequences (16).

Cell Extraction

Cells were harvested in extraction buffer (1% Triton X-100,

50 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 7.5) con-taining protease inhibitor cocktail (Roche, Mannheim,Germany) following two washes with ice-cold PBS. Thelysates were centrifuged at 10 000 g, 4�C for 10 min in a

microcentrifuge. The supernatants were normalized for pro-tein concentrations using a BCA protein assay kit (Pierce,Rockford, IL, USA). Bovine serum albumin was used as a

standard.

Grading Pigmentation

The cell pellets collected from above cell extractions werevisually inspected and graded from minus (no pigmentation)

to 4+ (pigmentation comparable with wild-type melano-cytes). The pigmentation grading of cell pellets is shown inFig. 1.

Western Blot Analysis

Proteins (20 lg for each lane) were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto membranes (Immobilon-P;

Millipore, Waltham, MA, USA).

Glycosidase Analysis

Glycosidase digestion was based on the protocol from New

England Biolabs (Beverly, MA, USA). Protein extract(20 lg) was denatured in 0.5% SDS, 1% b-mercaptoethanolat 100�C for 10 min. One-tenth volumes of each 0.5 Msodium phosphate (pH 7.5) and 10% NP-40 were added.

N-Glycosidase F (2 ll 500 U/ll) (New England Biolabs) or

Fig. 1. Effects of vacuolar-type (H+)-ATPase (V-ATPase) inhibitorsand ionophores on melan-p1 cells. Melan-p1 cells were treated with theindicated compounds for 48 h. Cell extracts were collected and digestedwith N-glycosidase F or endoglycosidase H and subjected to immuno-blotting analysis. BAF, bafilomycin A1; IBMX, 3-isobutyl-1-methylxan-thine.

Pigment Cell Res. 17, 2004 37

Endoglycosidase H (2 ll 5 mU/ll) (Boehringer, Mannheim,Germany) was used for digestion at 37�C for 1 h. Thereaction was terminated with SDS sample-loading buffer and

boiled at 100�C for 2 min.

Electron Microscopy

Adherent cells were fixed in wells with half-strength Kar-novsky’s fixative (17) in 0.2 M sodium cacodylate buffer at

pH 7.2 for 30 min at room temperature. The cells werewashed three times in buffer and treated with 1% osmoniumtetroxide containing 1.5% potassium ferrocyanide (18) for

30 min. The cells were washed, stained en bloc with 0.5%uranyl acetate for 30 min, dehydrated, and embedded inEponate 12. An area of each culture was cut out of the Eponcast, mounted on Epon pegs, and sectioned on an RMC MT

6000-XL ultramicrotome (RMC Products, Boeckeler Instru-ments, Tucson, AZ, USA). Ultrathin sections were thenstained with aqueous solutions of uranyl acetate (2%) and

lead citrate (0.3%) for 15 min each and then viewed andphotographed in a JEOL JEM-100CX transmission electronmicroscope (JEOL USA, Peabody, MA, USA). (All tissue

processing supplies were purchased from Ted Pella, Inc.,Tustin, CA, USA.)

RESULTS

Bafilomycin A1 and Monensin Alter Tyr Processing

Patterns in Melan-p1 Cells

Monensin and bafilomycin A1 induced melanin synthesis inmelan-p1 cells (Fig. 1). The degree of melanization of cells

treated with those compounds at the indicated concentrationis compatible with pigment levels in wild-type melan-a cells(data not shown). Two Tyr bands are observed upon

immunoblotting in melan-p1 cells. The lower band issensitive to both endoglycosidase H and N-glycosidase Fdigestion, indicating that this form of Tyr contains highmannose glycan and is retained in the ER (3). It is not

processed by Golgi enzymes and represents a non-processedimmature misfolded form of Tyr. The upper band is resistantto endoglycosidase H, but sensitive to N-glycosidase F,

indicating that it is processed by Golgi enzymes andrepresents a mature form of Tyr.

The mature form of Tyr increased 7–8-fold after treatment

with 20 nM bafilomycin A1 for 48 h with a concomitantdisappearance in the ER-retained Tyr. By contrast, monensinhad little effect on the level of mature Tyr at 4 lM but

increased the level of a 66 kDa band, which migrated at aposition similar to ER-retained Tyr. However, this 66 kDaband was partially resistant to endoglycosidase H digestionand appeared as a smear running from 66 to 56 kDa after

digestion. Thus, despite a similarity in apparent molecularweight, the 66 kDa Tyr in monensin treated melan-p1 cells isnot equivalent to the 66 kDa ER-retained Tyr observed in

untreated cells. The high mannose glycans of Tyr are partiallyprocessed by Golgi enzymes upon treatment with 4 lMmonensin. The resultant Tyr is a partial Golgi-processed Tyr.

We also examined the effects of IBMX, an inhibitorof cyclic adenosine monophosphate (cAMP) and cyclic

guanidine monophosphate (cGMP) phosphodiesterases, onmelan-p1 cells. We found that IBMX can increase melaninsynthesis in melan-a cells, but not in melan-p1 cells (data not

shown). It is thought that the melanogenic effects of IBMXare mediated through an increase in cAMP or cGMP (19).The levels of both mature Tyr and ER-retained Tyr in melan-p1 cells increased proportionally upon IBMX treatment.

Thus, IBMX appears to work at a transcriptional ortranslational level, boosting the expression of Tyr, but doesnot correct the Tyr defects in melan-p1 cells.

Induction of Melanin Synthesis in Melan-p1 Cells

Corresponds with Diminution of the Level of ER-Retained

Tyr, but not with Level of Tyr Protein

We examined the processing of Tyr in cells treated with

increasing doses of bafilomycin A1 and monensin (Figs 2 and3b). In addition, we also examined the response to thecysteine protease inhibitor E64 as we have shown previously

that E64 can diminish the secretion of Tyr into the culturemedium by melan-p1 cells (6). Unlike bafilomycin A1, E64did not induce melanin formation after 48 h incubation as

previously reported (6). However, E64 increased the level ofmature Tyr by 7–8-fold at 25 lM, an increase comparablewith that induced by 10–20 nM bafilomycin A1 (Fig. 2).

However, the level of ER-retained Tyr did not change upontreatment with E64.

Bafilomycin A1 increased the level of mature Tyr in a dose-dependent fashion (Fig. 2). However, despite similar levels of

mature Tyr in melan-p1 cells treated with 5–20 nM bafilo-mycin A1, no melanin synthesis is observed in melan-p1 cellstreated with 5 nM bafilomycin A1. While a slight drop in the

level of ER-retained Tyr was seen in cells treated with 5 nMbafilomycin A1, ER-retained Tyr disappeared upon treat-ment with 10 and 20 nM bafilomycin A1. When the results

with bafilomycin A1 and E64 are taken together, it is evidentthat melanin formation in melan-p1 cells is most closely cor-related with the conditions leading to the loss of ER-retainedTyr, not simply with the amount of mature Tyr.

We did not observe a synergistic effect between E64 andbafilomycin A1 (Fig. 3A). Addition of 25 lM E64 did notreduce the dose concentration of bafilomycin A1 required for

melanin synthesis in melan-p1 cells despite causing a strongincrease in levels of mature Tyr. No melanin was producedafter treatment with 3 nM bafilomycin A1 and 25 lM E64.

Fig. 2. Dose response of melan-p1 cells treated with bafilomycin A1 orE64. Melan-p1 cells were treated with bafilomycin A1 or E64 atincreasing concentrations for 48 h. Tyrosinase protein was visualizedby immunoblotting. Melanin formation was graded visually with 4+representing the amount of pigment formation in wild-type melanocytes.

38 Pigment Cell Res. 17, 2004

Melanin was only formed when the bafilomycin A1concentration was increased to 10 nM, with concomitant

loss of ER-retained Tyr.Melanin production was observed in melan-p1 cells treated

with 0.1 lM monensin and the amount of melanin produced

increased with higher concentrations (Fig. 3B). While the66 kDa Tyr band remained sensitive to endoglycosidase H at0.03 lM monensin, it became partially resistant at 0.1 lM

monensin, consistent with Golgi processing. The initiation ofmelanin production in melan-p1 cells treated with monensincorresponds well with the formation of this partial Golgi-processed Tyr. This partial Golgi processed Tyr differs from

mature 70 kDa Tyr only in its glycan composition asN-glycosidase F digestion reduced both bands to a 56 kDaband. Thus, the exact glycan composition does not appear to

be essential for melanin synthesis to occur in melan-p1 cells.

Melanin Deposition and Melanosomal Maturation in

Melan-p1 Cells Treated with Bafilomycin A1 and Monensin

As bafilomycin A1 and monensin target broad groups ofsubcellular compartments including the ERGIC (20), the

Golgi (13), and endosomal vesicles (21), we investigated thesubcellular site of melanin deposition in melan-p1 cells treatedwith those compounds.

As reported previously (10), mature melanosomes were not

observed in melan-p1 cells (Fig. 4). By contrast, after treat-ment of melan-p1 cells with bafilomycin A1 and monensin for48 h, stage III and IV mature melanosomes were induced

(Fig. 4). Thus, functional Tyr was distributed to melano-somes after bafilomycin A1 and monensin treatment.

Although melanin was primarily deposited in melano-

somes upon treatment with 50 nM bafilomycin A1, twoaberrations were seen in comparison with melan-a cells.First, many melanosomes appeared to be elongated andacquired �cucumber� shapes. Secondly, some endosome-like

structures also contained melanin (Fig. 4).Melanosomes from melan-p1 cells following monensin

treatment closely resembled those in melan-a cells (Fig. 4).

They exhibited an oval shape and correct size. Melanindeposition in endosome-like structures was not observed.However, large vacuoles were seen in cells treated with

monensin. Monensin has been previously shown in non-melanocytic cells to induce formation of swollen vesiclesthought to derive from dilated Golgi cisternae (13).

DISCUSSION

Multiple Tyr defects are observed in melan-p1 cells including

ER retention (3, 4, 6). Tyr exhibits an unusually longmaturation time in the ER. Branza-Nichita et al. (22)reported that Tyr in mouse B16 melanoma cell spent

approximately 3 h in interaction with calnexin, an ERchaperone protein, in contrast to 1 h interaction betweenan average glycoprotein and calnexin. Tyr undergoes mat-

uration much more slowly than Tyrp1 although the twoproteins share about 40% identity (23). Toyofuku et al. (24)

Fig. 3. Dose response of melan-p1 cells treated with bafilomycin A1 andE64 or monensin. (A) Melan-p1 cells were treated with 25 lM E64 andincreasing dose of bafilomycin A1. (B) Melan-p1 cells were treated withincreasing doses of monensin. The cell extracts were digested withendoglycosidase H or N-glycosidase F and subjected to immunoblotting.

Fig. 4. Electron microscopy analysis of melanin deposition in melan-p1 cells treated with bafilomycin A1 and monensin. Upon treatment with 50 nMbafilomycin A1 or 1 lM monensin for 48 h, mature melanosomes were observed. In addition, melan-p1 cells treated with bafilomycin A1 exhibitelongated melanosomes (arrows). Some endosome like structures (arrow head) also contain melanin. Bars, 1 lM.

Pigment Cell Res. 17, 2004 39

employed COS7 cells to study calnexin effects on Tyractivity. They found that cotransfection of calnexin andTyr increased Tyr activity by twofold over that observedupon transfection with Tyr alone.

The most common Tyr mutations are associated with ERretention of the protein, presumably the result of misfolding(25–27). For example, the temperature-sensitive Tyr mutant

R402Q can exit the ER at 31�C, but accumulates in the ER at37�C (26). In addition, substrates DOPA and tyrosinepromote Tyr exit from the ER and are associated with

induction of Tyr activity and melanin synthesis in amelanoticmelanoma cells (28). Proper ER processing of Tyr is crucialfor the subsequent enzymatic activity and subcellular local-

ization of the protein.As melan-p1 cells contain a significant amount of mature

Tyr as detected by immunoblotting (Fig. 1), it remains to beelucidated why this mature Tyr does not contribute to

melanin synthesis in these cells. Our current studies as well asothers suggest that this type of mature form of Tyr representsa �short-lived� form of Tyr (4). The majority of this Tyr fails

to reach melanosomes and, instead, is cleaved and secreted tothe extracellular media (4, 6). Thus, the mature Tyr inuntreated melan-p1 cells most likely represents a misfolded

Tyr, which is either not functional in vivo, travels to thewrong subcellular site, or both.

In the current study, we explored the melanogenic effects ofbafilomycin A1 and monensin on melan-p1 cells. The effects

of bafilomycin A1 on Tyr in melan-p1 cells appear to betwofold and are concentration dependent. Bafilomycin A1prevents the degradation of mature Tyr at concentrations

<10 nM. Bafilomycin A1 is known to raise the pH withinendolysosomal organelles. Ishidoh et al. (29) reported thatbafilomycin A1, which can increase lysosomal pH, caused the

degradation of cathepsin B, D, and L. We reported previ-

ously that the cathepsin family inhibitor, E64, could preventTyr secretion into the medium by melan-p1 cells (6). Thus,the effects of bafilomycin A1 on Tyr degradation in melan-p1cells may be mediated through a reduction in activity of

cathepsin proteases. However, this stabilization effect ofbafilomycin A1 toward Tyr contributes little to melaninsynthesis at least up to 5 nM concentration. Furthermore,

both E64 and IBMX raise the levels of mature Tyr withoutproducing melanin in melan-p1 cells. Bafilomycin A1 atconcentrations <10 nM either facilitates the production of

misfolded Tyr or prevents misfolded Tyr from degradation(see Fig. 5 for proposed model).

By contrast, bafilomycin A1 at concentrations >10 nM

promotes the release of Tyr from the ER to the Golgi, thusreducing the amount of Tyr trapped in the ER. BafilomycinA1 has been reported to block retrograde transport ofproteins from the pre-Golgi compartment to the ER (20). It

does not affect anterograde transport of protein from the ERto the Golgi. Halaban et al. (30) also reported that bafilo-mycin A1 can induce the release of Tyr from the ER and

promote melanin deposition in amelanotic melanoma cells.There appears to be a barrier in melan-p1 cells that preventstrafficking of ER-retained Tyr to the Golgi and this barrier is

bridged upon bafilomycin A1 treatment. Bafilomycin A1 is apotent V-ATPase inhibitor. It is reasonable to suggest that asubtype of V-ATPase exists in the ERGIC and bafilomycinA1 may raise the pH of the ERGIC. This results in proper

folding of Tyr and more effective processing from the ER tothe Golgi. This form of Tyr, after achieving proper folding, isable to reach melanosomes. This mechanism appears to be

the main contributor to the pharmacologically inducedmelanin synthesis in melan-p1 cells, as reduction in theER-retained Tyr in melan-p1 cells corresponds best with the

initiation of melanin synthesis.

Fig. 5. Proposed model for Tyr processingin melan-p1 cells treated with bafilomycinA1. A significant amount of Tyr is retainedin the ER of melan-p1 cells. Tyr that isable to reach the Golgi is misfolded andtravels to the cell membrane and endosomalorganelles, where degradation occurs. Lowconcentrations of bafilomycin A1 preventdegradation and result in the accumulationof misfolded Tyr. High concentrations ofbafilomycin A1 increase the pH of theERGIC, which enhances correct Tyr foldingand more effective ER–Golgi processing ofTyr. This form of Tyr reaches melanosomesand participates in the melanin synthesis.

40 Pigment Cell Res. 17, 2004

Monensin treatment resulted in a disappearance ofthe ER-retained Tyr and the accumulation of a par-tial Golgi-processed Tyr. Effects of monensin on the

preGolgi-compartment have been reported (31–33). Halabanet al. (30) proposed that monensin may raise the pH of thepre-Golgi compartment in amelanotic melanoma cells andallow ER-retained Tyr to pass through. A similar explan-

ation may also apply to melan-p1 cells upon treatment withmonensin. Similar to bafilomycin A1, monensin may raisethe pH of the ERGIC and allow the release of Tyr into the

Golgi for subsequent processing. Unlike bafilomycin A1,monensin also targets the Golgi and TGN by disruptingNa+/H+ exchange and may prevent Tyr from becoming

fully processed. However, this partial Golgi-processed Tyr isstill functional and can reach melanosome to induce melaninformation.

Our previous data showed that Tyr could be restored to adispersed punctate distribution from a perinuclear locationupon treatment of melan-p1 cells with bafilomycin A1 orNH4Cl (3). We have now further investigated the site of

melanin deposition in treated melan-p1 cells. Electronmicroscopy (EM) analysis reveals that melan-p1 cells con-tained many mature melanosomes after treatment with

bafilomycin A1 or monensin, indicating that each compoundcan restore Tyr trafficking to melanosomes, and melaninformation is mainly contained within melanosomes.

Elongated melanosomes and melanin deposition in endo-some-like structures were observed in melan-p1 cells treatedwith 50 nM bafilomycin A1 (Fig. 4). The precise cause for theobservation is not known, although one may speculate that it

maybe the result of neutralization of pH of those organelles orin vesicles that must fuse with them. A higher pH subcellularenvironment may cause Tyr to partially sort to endosome-like

structures and a low internal pH may be an important factorin regulating the size and shape of melanosomes.

Our current studies show that bafilomycin A1 and

monensin induce melanin synthesis in the absence of p byfacilitating the release of Tyr from the ER to the Golgi. Thisenables proper Tyr folding and sorting to the melanosomes,

where melanin synthesis occurs.

Acknowledgement – This work was supported by PHS grant AR41880(SJO) and by funding from the Anaderm Research Corporation.

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