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Molecular basis of the extreme dilution mottled mouse mutation: a combination of coding and non-coding genomic alterations

Alfonso Lavado1, Concepción Olivares2,3, José Carlos García-Borrón2, Lluís Montoliu1,*

(1) Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular and Cellular Biology,Campus de Cantoblanco, C/ Darwin 3, 28049 Madrid (Spain)

(2) Departament of Biochemistry and Molecular Biology and Immunology, School of Medicine,University of Murcia, Apto 4021, Campus Espinardo, 30100 Murcia (Spain)

(3) Present address: Laboratoire d’ Enzymologie et Biochimie Structurales (CNRS), 1 Avenue de laTerrasse. 91198 Gif-sur-Yvette, France.

Running Title: molecular basis of the extreme dilution mottled mouse mutant

(*) Author for correspondence: Lluís Montoliue-mail: montoliu@cnb.uam.es

tel. +34-915854844 / fax. +34-915854506

SUMMARYTyrosinase is the rate-limiting enzyme inmelanin biosynthesis. It is a N-glycosylated,copper-containing transmembrane protein,whose post-translational processing involvesintracytoplasmic movement from theendoplasmatic reticulum (ER) to the Golgi and,eventually, to the melanosome. The expressionof the tyrosinase (Tyr) gene is controlled byseveral regulatory regions including a LocusControl Region (LCR) located 15 kb upstreamfrom the promoter region. The extreme dilutionmottled mutant mice (Tyrc-em) arosespontaneously at the MRC Institute in Harwell(UK) from a chinchilla-mottled mutant (Tyrc-m)stock, whose molecular basis corresponds to arearrangement of 5’ upstream regulatorysequences including the LCR of the Tyr gene.Tyrc-em mice display a variegated pigmentationpattern in coat and eyes, in agreement with theLCR translocation, but also show a generalisedhypopigmented phenotype, not seen in Tyrc-m

mice. Genomic analyses of Tyrc-em mice showed aC1220T nucleotide substitution within the Tyrencoding region, resulting in a T373I aminoacid change, that abolishes a N-glycosylationsequon located in the second metal ion bindingsite of the enzyme. Tyrosinase from Tyrc-em

displayed a reduced enzymatic activity in vivoand in vitro, compared to wild-type enzyme.Deglycosylation studies showed that the mutantprotein has an abnormal glycosylation pattern

and is partially retained in the ER. We concludethat the phenotype of the extreme dilutionmottled mouse mutant is caused by acombination of coding and non-coding genomicalterations resulting in several abnormalitiesthat include suboptimal gene expression,abnormal protein processing and reducedenzymatic activity.

INTRODUCTIONTyrosinase (Tyr1; monophenoldihydroxyphenylalanine: oxygen oxidoreductase;EC 1.14.18.1) (1, 2) is the rate-limiting enzyme inmelanogenesis and catalyses the conversion of L-tyrosine into L-dopaquinone in the presence of L-3,4-dihydroxyphenylalanine (L-DOPA) (3). Tyr isa transmembrane glycoprotein that undergoes acomplex post-translational processing beforereaching the melanosome. In mice, Tyr processingincludes: N-glycosylation in at least four out of sixavailable glycosylation sites (4); translocation fromthe endoplasmic reticulum (ER) to the Golgiapparatus; binding of the copper cofactor to thetwo sites known as CuA and CuB, and finallysorting to the melanosomes in its finalcatalytically-active conformational state (5, 6). Therelevance of Tyr post-translational processing isenhanced by the recent consideration of sometypes of oculocutaneous albinism (OCA) as ERretention diseases. Oculocutaneous albinism type 1(OCA1) is an autosomal recessive disease resultingfrom mutations in the human tyrosinase (TYR)

JBC Papers in Press. Published on November 30, 2004 as Manuscript M410399200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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gene (7, 8), usually associated with abnormalprocessing of the enzyme (9-12). Relatedpigmentary disorders associated with mutations inother genes, such as OCA2 (13, 14), OCA3 (12),OCA4 (15) and more complex diseases asHermansky-Pudlak and Chediak-Higashisyndromes (16, 17, 18) can also be accounted forby abnormalities in processing or intracellulartrafficking of the corresponding proteins.Mouse models for nearly all human pigmentarydisorders have been reported (reviewed in 19;listed in 20, 21). Mutations in the mouse tyrosinase(Tyr) gene are mainly associated with alterations inthe coding sequence, as in the albino (Tyrc) (22),himalayan (Tyrc-h) (23), dark eyed (Tyrc-44H) (24)and platinum (Tyrc-p) (25) alleles. Some of thesemutations in the Tyr gene are associated not onlywith low enzymatic activities but also withprocessing or trafficking defects (6, 11, 12, 25).The Tyr gene is exclusively expressed in two celltypes: melanocytes, derived from migrating neuralcrest cells, that eventually colonise iris, choroid,skin, and cochlea, and retinal pigmentedepithelium (RPE) cells, derived from the optic cup(26, 27). In mice, the expression of Tyr is tightlyregulated by a combination of proximal promoterelements (28-31) and distal elements, mostremarkably a locus control region (LCR), located15 kb upstream from the transcription start site(32-36). The presence of a LCR was inferred fromthe analyses of artificial chromosome-typetransgenes in mice harbouring the entire Tyrexpression domain (37, 38), and from previousstudies on the molecular basis of the chinchillamottled mutant allele of the Tyr gene (Tyrc-m) (39-41). Tyrc-m mice displayed a variegatedpigmentation in the coat, RPE cells and choroid(39, 40). This phenotype was correlated with agenomic rearrangement of ~30 kb 5’ upstream Tyrregulatory sequences, which included the LCR(41). Interestingly, the subsequent analysis oftransgenic mice carrying large Tyr genomicconstructs, in which the LCR has beenexperimentally deleted, showed most similarvariegated phenotypes (42, 43).The extreme dilution mottled (Tyrc-em) mutationarose spontaneously at the MRC MammalianGenetics Unit in Harwell (UK) from a chinchillamottled mouse stock (Peter Glenister, personalcommunication). Tyrc-em mice display a variegatedpattern of pigmentation in coat, but also an overall

hypopigmented phenotype, as compared tochinchilla mottled mice, from which theyoriginated, thus suggesting the presence ofadditional mutations in the Tyr gene. Themolecular basis of the extreme dilution mottledmutation was not known. We decided toinvestigate this Tyr allele as we considered it couldcontribute to the understanding of the Tyr generegulation, including LCR function. We haverescued Tyrc-em mice from frozen embryos andperformed a series of phenotypic, genomic andbiochemical analyses that have resulted in thedescription of the molecular and functional basisof the extreme dilution mottled mutation.

EXPERIMENTAL PROCEDURESAnimals - The following types of mice were usedin this study: heterozygous wild-type pigmentedYRT2 YAC-tyrosinase transgenic mice (YRT2/- ;Tyrc/Tyrc) (line #1999, 37), heterozygous YRT4YAC-tyrosinase transgenic mice (YRT4/- ;Tyrc/Tyrc) (line #2138, 34, 42), homozygous orheterozygous extreme dilution mottled (Tyrc-em)and chinchilla mottled mutant mice (Tyrc-m) (39,40, 41) and albino outbred NMRI (HarlanInterfauna Iberica, S.L., Barcelona, Spain) mice(Tyrc/Tyrc). Tyrc-em and Tyrc-m mice were rescuedfrom frozen embryos (embryos kept frozen since1984) at the Frozen Embryo and Sperm Archive(FESA) of the MRC Mammalian Genetics Unit, inHarwell (UK) and sent to CNB (Madrid, Spain)with the following initial genotypes: Tyrc-em/Tyr+

and Tyrc-m/Tyrc-m; a/a; Ednrbs/Ednrb+, respectively.Thereafter, extreme dilution mottled and chinchillamottled mutant mice, as well as YRT2 and YRT4transgenic mice, were bred and have beenmaintained in albino outbred NMRI geneticbackground. Histological analysis of adult mouseretinae was performed as reported (42). Allexperiments complied with local and Europeanlegislation concerning vivisection and theexperimentation and use of animals for researchpurposes.

Quantification of melanin and tyrosinaseenzymatic activity assays - Melanin contentswere measured by spectrophotometry as describedpreviously (44). Two types of tyrosinase assayswere performed in whole eye extracts followingdescribed procedures (45, 46). The first oneconsists of a tyrosine hydroxylase activity

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determination by a radiometric method based onthe release of tritiated water from labelled L-[3,5-3H]-Tyrosine. The second assay measures melaninformation from L-[14C(U)]-Tyrosine, and thereforeprovides an estimate of rate of the completemelanogenic pathway. The tyrosine hydroxylaseactivity of Tyr in whole-cell extracts wasdetermined using the same radiometric methoddescribed above, except that shorter incubationtimes (1 hour instead of 14-16 hours) and smallervolumes of extract (usually 5 µl of the cell lysatesinstead of 25 µl, used for whole eye extracts) wereemployed. One unit was defined as the amount ofenzyme catalysing the hydroxylation of 1 µmol ofL-tyrosine/min, in the presence of a 50 mMconcentration of the substrate and 10 mM L-DOPA as cofactor (47). For statistical analyses,where indicated, t-Student paired tests wereapplied and statistical signification indicated asfollows: * p<0.05; ** p<0.01; *** p<0.001(StatView, SAS Institute, Inc., Cary, NC).

Southern blot analyses - For high-resolutionSouthern blot analysis, megabase-size genomicDNA from mouse cells was prepared fromsplenocytes following described procedures (48).EcoR I and Apa I digestions (0.5 U / ml) were runat 37oC for 12-14 hours on agarose plugs(Seaplaque LMP, Cambrex Bio Science Rockland,Inc., Rockland, ME) containing mouse genomicDNA. Plugs were resolved by horizontal gelelectrophoresis in a 20 x 25 cm gel of 0.5% (w/v)Seakem Gold Agarose (Cambrex Bio ScienceRockland, Inc.), 1x TAE buffer (40 mM Tris-Acetate, 2 mM EDTA), at 3 V/cm, 4oC for 16hours. The gel was transferred onto a Hybond-Nnylon membrane (Amersham Biosciences EuropeGmbH, Barcelona, Spain) by capillary blotting andhybridised with a random-primed [α32P]-dCTP-labelled D probe (904 bp LCR internal probe,nucleotide positions +8574 to +9478, GenBankAF364302, 35) in hybridisation buffer (0.25 MNa2HPO4 pH 7.2, 7% (w/v) SDS, 1% (w/v) BSA)for 16 hours at 65oC, washed at 65oC in 20 mMNa2HPO4 pH 7.2, 1% (w/v) SDS, 1 mM EDTA pH8.0 and, finally, autoradiographed and/or analysedwith a PhosphorImager (Molecular Dynamics,Amersham Biosciences).Standard Southern blot analyses were carried outusing previously described procedures (34). Inbrief, 15-20 µg of genomic DNA from mouse tail

biopsies were digested with EcoR I or Hind III(Roche Diagnostics, S.L., Barcelona, Spain),fractionated by horizontal electrophoresis in a 1%(w/v) agarose (Invitrogen S.A., Barcelona, Spain)gel, transferred by capillary blotting onto Hybond-N nylon membranes (Amersham Biosciences) andsubsequently hybridised with a full-length mouseTyr cDNA probe (pmcTyr1, 28).

Rescue of the Tyrc-em cDNA and preparation of aTyrc-em expression construct - Several overlappingfragments comprising the entire Tyrc-em codingregion were obtained by PCR amplification ofgenomic DNA with Vent DNA polymerase (NewEngland Biolabs, Beverly, MA) or by RT-PCR,from RNA extracted from eyes (Rneasy Mini Kit,Qiagen GmbH, Hilden, Germany), using specificoligonucleotides designed according to reportedmouse Tyr cDNA sequences (GenBank X12782and M24560). PCR products were cloned intopCRII T/A vector (Invitrogen), sequenced andanalysed (alignments) using MacVector (Accelrys,Inc., San Diego, CA). Detailed description ofoligonucleotides and plasmids used is availableupon request. A full-length Tyrc-em cDNA wasreconstituted, entirely sequenced (GenBank2

AY526904) and cloned into a pcDNA3 expressionvector (Invitrogen). DNA sequences were obtainedand confirmed from three independent Tyrc-em

homozygous mutant mice. The Tyr+ expressionconstruct, as well as those encoding the Tyr∆6 andTyrMeB mutant variants, all generated in pcDNA3,have been previously described (47).

Culture and transient transfection ofmammalian cells - Human embryonic kidney(HEK) 293T cells (American Type CultureCollection CRL number 1573) were cultured at37oC / 5% CO2 in 6-well plates (Falcon, BectonDickinson Labware Europe, Le Pont De Claix,France) with RPMI-1640 medium (Invitrogen),10% (v/v) fetal calf serum (Invitrogen), 100units/ml penicillin and 100 µg/ml streptomycin(Invitrogen). Cells were transiently transfectedwith Lipofectamine 2000 (Invitrogen), accordingto manufacturer’s recommendations, and harvested24 hours after transfection. Whole-cell crudeextracts were prepared in 100 µl of 10 mM sodiumphosphate buffer pH 6.8, 0.1 mM EDTA pH 8, 1%(v/v) Igepal CA-630, 0.1 mM PMSF (Roche) aspreviously reported (47). The protein content of

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the extracts was determined by the bicinchoninicacid method, using bovine serum albumin asstandard.

Deglycosylation analysis - Endo H or Endo Fdigestions were made as described previously (47).In brief, 8 µg of total protein were incubated at37oC for 3 hours in the presence of 20 mU of EndoH or 4 U of Endo F (Roche) in 50 mM sodiumphosphate buffer pH 7.0 with 10 mM EDTA, 0.1%(w/v) SDS. Analytical SDS-PAGE was performedin 12% (v/v) acrylamide gels as described (47, 49).Transfer to a polyvinylidene difluoride membrane(Immobilon-P, Millipore Iberica S.A., Madrid,Spain) was undertaken in a semidry unit (Bio-RadLaboratories, Hercules, CA). Immunodetection oftyrosinase was performed with rabbit polyclonalαPEP7 (a generous gift from Dr. V. Hearing,National Institutes of Health, Bethesda, MD) (50)following previously published procedures (51).Staining and detection were done with the ECLPlus chemiluminescent substrate (AmershamBioscience).

Immunocytochemistry - HEK293T cells weretransiently transfected with the Tyr+, TyrMeB, Tyr∆6

(47) and Tyrc-em cDNA constructs, cloned inpcDNA3 vector, as described above.Immunocytochemistry was performed aspreviously described (12). Briefly, 24 h posttransfection, cells were fixed in 4% (w/v) PFA for15 min at 25oC and then permeabilized withmethanol for 20 min at 4oC. After blocking with10% (v/v) fetal calf serum in PBS for 1 h at 25oC,cells were incubated with 1:500 mouse monoclonalantibody against KDEL (StressgenBiotechnologies, Inc., San Diego, CA) (a generousgift from Dr. V. Hearing, National Institutes ofHealth, Bethesda, MD) in 2% (v/v) fetal calf serumin PBS overnight at 4oC. Cells were washed threetimes in PBS and then incubated with 1:500αPEP7 in 2% (v/v) fetal calf serum in PBS (50),for 1 h at 25oC. Appropriate secondary antibodieswere used to reveal specific signals: Alexa 594-labelled goat anti-mouse (dilution, 1:500) andAlexa 488-labelled goat anti-rabbit (dilution,1:500) (Molecular Probes, Invitrogen), for 1h at25oC. Nuclei were counterstained with 1:200 TO-PRO3 in PBS (Molecular Probes). All preparationswere examined in an Axiovert 200 confocalmicroscope (Carl Zeiss AG, Oberkochen,

Germany) and images were processed with LaserSharpe 2000 program (Bio-Rad Laboratories).

RESULTSPhenotypic analysis of extreme dilution mottledmutant miceThe phenotype of adult heterozygous extremedilution mottled mice (Tyrc-em/Tyrc) wascharacterised by ruby eyes and a very faintlypigmented, almost albino coat. Homozygousextreme dilution mottled mice showed darker eyes,and variegated light-grey coat, with morepigmentation than observed in heterozygousanimals. Both heterozygous and homozygousextreme dilution mottled mice showed lesspigmentation than chinchilla mottled (Tyrc-m) mice(41), from which the Tyrc-em arose (Figure 1A).In the eye, both heterozygous and homozygousextreme dilution mottled mice exhibited avariegated pigmentation pattern at the retinalpigment epithelium (RPE) cells and irises (Figure1B-G), similar but much lighter to that previouslydescribed of adult chinchilla mottled (Tyrc-m) mice(41) (Figure 1H-J). In contrast to chinchillamottled mice, the choroid of extreme dilutionmottled mice was weakly pigmented (Figure 1K-M).

Tyrosinase activity and melanin content in theeyes of extreme dilution mottled mutant miceWe measured the Tyr enzymatic activity ofheterozygous extreme dilution mottled (Tyrc-

em/Tyrc) mutant mice in crude protein eye extractsand compared it with the corresponding enzymaticactivities of albino mice (Tyrc/Tyrc, NMRI),heterozygous chinchilla mottled (Tyrc-m/Tyrc) mice(39-41), YRT4 and YRT2 transgenic mice (34,37). YRT2 animals are indistinguishable fromwild-type pigmented mice (52, 53), whereas YRT4mice display a variegated pigmentation phenotypein the eye and coat, associated with theexperimental deletion of the locus control region(LCR) in the YAC-tyrosinase transgene (42).The tyrosine hydroxylase and melanogenicactivities of Tyr in extreme dilution mottled micewere intermediate between those of control albinoNMRI and wild-type pigmented YRT2 transgenicmice, but indistinguishable from those observed inchinchilla mottled or YRT4 transgenic mice(Figure 2A-B). Whole eye melanin contents inextreme dilution mottled mice were lower than in

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any other pigmented mouse analysed, and similarto albino NMRI mice (Figure 2C).

Genomic analysis of the Tyrc-em locusTo gain insight into the potential alterationsaffecting the Tyrc-em locus we performed a series ofgenomic analyses (Figure 3). First, we investigatedusing Southern blot analysis the presence andendogenous location of the LCR element in theTyrc-em mutant allele on homozygous extremedilution mottled genomic DNA samples digestedwith restriction enzymes EcoR I and Apa I, usingan internal LCR-specific probe (probe D, Figure3A, 35). The expected 3.7 kb EcoR I DNAfragment containing the LCR element (32) wasfound in all mice analysed (Figure 3B). Further,we observed that the Tyrc-em LCR was locatedapproximately at -40 kb from the transcription startsite, at a similar distance as previously observed inchinchilla mottled mice, further upstream from itsendogenous normal position in wild-type mice,found approximately at –15 kb (35, 40) (Figure3A).Second, we studied the entire genomic structureand integrity of the Tyr locus in the Tyrc-em mutantallele by Southern blot analysis, using a full-lengthTyr cDNA probe (pmcTyr1, 28) and two analyticalrestriction enzymes: EcoR I and Hind III (28, 34).No obvious differences were observed in theDNA-banding pattern of the Tyr gene between themutant Tyrc-em and Tyrc-m alleles, compared withthe expected pattern of wild-type Tyr+ locus(Figure 3A, 3C).Third, we obtained the entire coding sequence ofthe Tyr gene in the extreme dilution mottled mutantmice by PCR and RT-PCR, using a number ofspecific oligonucleotides. Several overlappingDNA fragments comprising the whole Tyrc-em

cDNA were obtained, aligned and a full-lengthcDNA sequence was eventually assembled(GenBank AY526904). The analysis of the Tyrc-em

cDNA sequence showed a single nucleotidemutation, cytidine (C) to thymidine (T), at position+1220, according to wild-type Tyr+ cDNA(GenBank X12782). This point mutation wouldresult in an amino-acid substitution, threonine (T)to isoleucine (I), at position 373 in the predictedprotein sequence, as compared to wild-type tyrprotein (SwissProt P11344).

Processing and deglycosylation analysis of Tyrfrom the extreme dilution mottled miceTyr amino acid position 373 is located within thesixth potential N-glycosylation sequon of theenzyme, within the CuB binding site (47). Thisglycosylation sequon is conserved in mammaliantyrosinases (6), is glycosylated in Tyr (4, 47), andits occupancy has been reported to correlate withcofactor binding, and acquisition of full enzymaticactivity (4, 47), as well as proper processing. Toinvestigate whether the T373I mutation impairs theprocessing and glycosylation of Tyr in extremedilution mottled mutant mice (Tyrc-em), wetransiently transfected HEK293T cells with wild-type or Tyrc-em expression constructs followed byWestern blot analysis of detergent-solubilizedextracts with a Tyr-specific antibody (αPEP7, 50,51). HEK293T cells (54) have been shown toreproduce the normal Tyr post-translationalprocessing observed in B16 mouse melanoma cells(47). For comparison, we also analyzed twotyrosinase mutants, Tyr∆6 and TyrMeB. Tyr∆6 bears aN371Q conservative substitution predicted toabolish the same 371NGT373 glycosylation sequonas the extreme dilution mottled mutation. TyrMeB isa chimeric construct consisting of the completeTyr sequence except for the replacement of theentire CuB site (from residue His363 to residueHis390) by the homologous fragment of thetyrosinase-related protein 1 (Trp1). Thepreparation and characterization of these mutantshas been described (47). Tyr∆6 is partially activeand is not glycosylated in one glycosylationsequon, whereas TyrMeB is totally inactive,underglycosylated in more than one site, andretained in the ER as judged by itsendoglycosydase digestion pattern (47). Westernblot analysis of extracts from HEK293T cellstransfected with the wild-type Tyr expressionconstruct, showed a major broad band (apparentmolecular weight, Mr, ~78 kDa) and faster-migrating bands (Mr ~69 kDa and lower),corresponding to complete and partiallyglycosylated forms, respectively (Figure 4A). Asexpected, the electrophoretic mobility of the Tyr∆6

and Tyrc-em mutants was very similar, and higherthan for the corresponding forms of wild-type Tyr.The mobility of the TyrMeB construct was evenhigher. This pattern is fully consistent withunderglycosylation in one site for the Tyr∆6 andTyrc-em mutants. Interestingly, the intensity of the

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signal in Tyrc-em samples was consistently lowerthan for the wild-type enzyme for comparableprotein loads (37 ± 6% of controls, n = 3),indicative of lower levels of the mutant enzyme(Figure 4B).The processing of Tyrc-em was further assessed invitro by means of glycosydase digestion withEndoH and EndoF. EndoH removes N-linled highmannose-type carbohydrates characteristic ofendoplasmic reticulum (ER)-resident, incompletelyprocessed forms of N-linked glycoproteins. Uponfurther processing at the medial Golgi theseproteins become resistant to EndoH. Hence,EndoH provides a way to distinguish immatureforms of Tyr from the mature enzyme. In turn,EndoF digests all forms of N-glycans,independently of their degree of processing, thusallowing for the estimation of the size of theprotein backbone (9, 11, 12, 47).The wild-type Tyr+ protein was largely resistant toEndoH digestion, thus suggesting a mature, post-ER conformation (Figure 4C). In contrast, thesensitivity to EndoH of Tyrc-em was higher,consistent with incomplete or abnormal processingand significant retention in the ER. Theelectrophoretic mobility of the single band presentafter EndoF-digestion, corresponding to thedeglycosylated protein backbone with an apparentMr of 55 kDa, was identical to wild-type. Takentogether, these data suggested that Tyrc-em is notproperly glycosylated and therefore is likelyretained, at least partially, in the ER.The potential retention of Tyrc-em in the ER wasfurther investigated by confocal microscopy(Figure 4D). HEK293T cells were transientlytransfected with wild-type Tyr, Tyrc-em, Tyr∆6 orTyrMeB and stained with the tyrosinase-specificαPEP7 antiserum (12, 50), and with the ER-specific KDEL antibody (12). In cells expressingthe wild-type form, no significant co-localisationof tyrosinase and ER-specific signals was detected,as reported for melanocytes. Conversely, extensiveco-localisation of TyrMeB and the ER-specificmarker was detected, consistent with previouslyreported biochemical analysis (47). Finally, alimited co-localisation of the Tyrc-em and Tyr∆6

mutants and the ER marker was seen.

Residual enzymatic activity of Tyrc-em

The occupancy of the sixth potential N-glycosylation sequon has been correlated with full

Tyr enzymatic activity (4, 47), but the residualactivity in different mutants where this site isnegated may be dependent on the particular aminoacid substitution considered (47). To determine theresidual activity of Tyrc-em, we measured in vitro,the Tyr enzymatic activity and kinetic constants ofthe mutant protein and compared them with thoseof wild-type Tyr+, using whole-cell extractsprepared from HEK293T transfected cells. TheTyrc-em mutant protein displayed higher KM andlower Tyr activity (~13% of wild-type) and Vmax,resulting in a catalytic efficiency (Vmax / KM)reduced almost 3-fold, as compared to wild-typeTyr+ (Table I).

DISCUSSIONThe LCR plays a fundamental role in regulatingthe expression of the Tyr gene in melanocytes andRPE cells (34, 42). The analysis of mice carrying aseries of Tyr mutant alleles or plasmid/YAC-Tyrtransgenes has allowed the characterisation of thisregulatory element (32-35, 40, 42, 55, reviewed in43). In chinchilla mottled mice, a likelytranslocation of 5’ upstream regulatory sequences,including the LCR, from -15 kb to approximately -40 kb from the Tyr transcription start site results ina variegated pigmented phenotype in the coat andRPE cells (40, 41). YRT4 transgenic mice,carrying a modified YAC-Tyr transgene devoid ofthe LCR, displayed a similar variegateddistribution of melanin in the coat and eye,although with a lower pigmentation level, ascompared to chinchilla mottled mice (34, 42). Theextreme dilution mottled mouse mutationoriginated from a chinchilla mottled mouse stockat the MRC-MGU (Harwell, UK), and ischaracterised by a variegated pigmented phenotypein the coat, iris and RPE cells, combined with ageneralised reduced pigmentation, compared toeither chinchilla mottled or YRT4 mice (Figure 1,34, 42). This enhanced hypopigmented phenotype,according to the differences observed betweenchinchilla mottled and YRT4 transgenic mice,suggested that at least two alternatives werepossible to explain the molecular basis of theextreme dilution mottled mouse mutation: (1) thepresence of an additional alteration in the LCR (orany regulatory element) within the Tyrc-em mutantallele, and/or (2) a mutation within the codingregion of the Tyrc-em mutant allele eventuallyresulting in a severely reduced Tyr enzymatic

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activity. To understand the basis for the extremedilution mottled mouse mutation, we decided toinvestigate these mutant mice with a variety ofapproaches, including detailed phenotypiccharacterisation, biochemical properties of themutant Tyr protein and exhaustive genomicanalysis of the Tyrc-em mutant locus.Firstly, we measured the observed decrease ofpigmentation in extreme dilution mottled mice byquantifying the Tyr activity and melanin content incrude eye extracts from adult heterozygous mice(Figure 2). Tyr enzymatic activities in crude eyeextracts of extreme dilution mottled mice were lowand intermediate between those of albino NMRImice and wild-type pigmented YRT2 transgenicmice, but comparable to those recorded inchinchilla mottled and YRT4 hypopigmentedmice. However, the melanogenic activity ofextracts from chinchilla mottled appearedsomewhat higher than that of extreme dilutionmottled mice. In keeping with this trend, weobserved a statistically significant decrease inocular melanin contents of extreme dilutionmottled mice, as compared to any other pigmentedmice analysed, and in particular with chinchillamottled mice. This slight discrepancy between therelative levels of Tyr activity measured in cellextracts and the melanin contents of the cells canbe explained by several factors. Indeed, theenzymatic activities measured in cell extracts maynot reflect perfectly the activity within the cell, orthe actual melanin formation potential, likely as aresult of altered subcellular location of the enzyme.This situation is not uncommon and is seen, forinstance, in tyrosinase-positive albinisms.Alternatively, the lower melanin contents mayreflect an actually reduced Tyr capacity to generatemelanin in these mutant mice. In this respect, it isworth noting that Tyrc-em carries a mutation in theCuB site, and that mutations in the metal bindingsites of Tyr are frequently associated with differenteffects on the various enzymatic activities of theprotein (6, 56).Next, we performed a systematic genomic analysisof the Tyrc-em locus, aiming to discover anysubsequent alterations present in the Tyrc-em mutantallele (Figure 3). First, the location of the LCR inthe Tyrc-em locus was found at a comparablegenomic position with regard to the Tyrc-m locus,from which it originated (Figure 3A, 3B). Thisrearrangement can mostly explain the variegated

pigmentation pattern observed in extreme dilutionmottled mice, as compared to the similar reportedphenotype in chinchilla mottled mice (39-41).Second, we assessed the integrity of the Tyr genein the Tyrc-em locus and did not find obviousdifferences in the expected DNA-banding pattern,as compared to either Tyrc-m or Tyr+ alleles. Third,we generated a full-length cDNA and entirelysequenced the coding region of the Tyrc-em locusthus detecting a single nucleotide substitution(C1220T) that would result in a predicted aminoacid T373I change. This non-conservativesubstitution abolishes the sixth potential N-glycosylation site, starting at position 371, andlocated within the CuB binding motif (6, 47).Changes at this position have been associated withOCA1 cases and aberrant processing of humanTYR (57, 58).Correlations between Tyr glycosylation,processing and intracellular traffic, and enzymaticactivity have been demonstrated (4), althoughseveral exceptions to this rule have also beenreported. For instance, Tyr protein variants with asimilar extent of processing and glycosylation butdisplaying different enzymatic activities have beendescribed (47). Therefore, we analyzed the effectsof this mutation on the glycosylation pattern,processing and enzymatic activity of the Tyrc-em

mutant, as compared with the wild type enzymeand two other mutants, Tyr∆6 and TyrMeB. In theTyr∆6 mutant, the sixth glycosylation sequon ismissing as a result of a N371Q substitution. TheTyrMeB quimeric construct is enzymaticallyinactive and underglycosylated in more than onesite (47). For this study, HEK293T cells wereselected as the most appropriate model system forthe transient expression of the different tyrosinaseforms. These cells share the same embryonicorigin as melanocytes (54) and express efficientlywild-type tyrosinase and various enzymaticallyactive mutants (47, 59). More importantly, theprocessing and glycosylation pattern of tyrosinaseand other melanogenic proteins like Trp1 seemsidentical in HEK293T cells and B16 mousemelanoma cells (47).The electrophoretic mobility of the Tyrc-em mutantprotein was identical to the Tyr∆6 mutant, andlower than the TyrMeB quimeric construct,underglycosylated in at least two sites. This is fullyconsistent with ablation of the sixth glycosylationsequon due to the observed T373I mutation,

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resulting in underglycosylation, as compared towild-type. Moreover, digestion with EndoHfollowed by Western blot showed that aconsiderable fraction (52 ± 7%, n = 3) of the totalTyrc-em protein present in crude extracts is sensitiveto the glycosydase, indicative of aberrant orincomplete processing and likely partial retentionin the ER. This possibility was further investigatedby immunocytochemistry, addressing the potentialco-localisation of Tyr and ER markers. In theseexperiments, no significant co-localization of wild-type Tyr and the ER marker was observed, furthersupporting the use of HEK293T cells as a suitablemodel for Tyr processing studies. On the otherhand, the TyrMeB mutant, which provided a positivecontrol for co-localisation, displayed a clear ER-retention phenotype consistent with its lack ofenzymatic activity and its full sensitivity to EndoH(47). The Tyrc-em protein and the Tyr∆6 mutantshowed a similar pattern, with limited butdetectable co-localisation with the ER marker inHEK293T transfected cells. Notably, in HEK293Tcells transiently expressing the various tyrosinaseforms, the levels of the Tyrc-em protein were lowerthan those of wild-type and Tyr∆6, and comparableto TyrMeB. This suggests a decreased intracellularstability of the protein that cannot be explainedexclusively by lack of occupancy of the sixthglycosylation sequon in Tyrc-em, since N-glycanaddition to this site is also abrogated in theTyr∆6mutant. Therefore, it would appear that thenon-conservative substitution in Tyrc-em induces aless stable conformation than the conservativemutation in Tyr∆6, in addition to the common effecton glycosylation of both mutations.The extreme dilution mottled mutant Tyr retainedsome measurable enzymatic activity, in contrast to

a related and previously reported T373K mutationin the human TYR enzyme. This conservation of ameasurable residual activity is consistent with thefinding of a fraction of the protein that appearsEndoH-resistant, and therefore fully processed.The retention of a sizeable residual enzymaticactivity is also consistent with the incomplete co-localization with ER markers, as opposed to thefully inactive TyrMeB protein. This partial activityof the Tyrc-em enzyme, together with the limited co-localisation with ER markers and with the partialsensitivity to EndoH, agrees with our previousobservations that glycosylation of the CuBacceptor sequon is not a “sine qua non”requirement for ER export, Cu binding andcomplete maturation of the enzyme. As opposed tothis situation, the T373K mutation in human TYRyields an enzyme which is fully retained in the ER,is not glycosylated in the equivalent sequon anddoes not have detectable Tyr activity (57, 58). Thisdiscordance can be explained again consideringthat the amino acid substitutions are very differentin mouse Tyrc-em and in human OCA-associatedT373K mutation.With all these data, we can conclude that thephenotype of the extreme dilution mice isgenerated by the combined effect of the LCRtranslocation (responsible for most of thevariegated pigmentation pattern observed) and asingle base pair mutation, C1220T, within thecoding sequence of the Tyr gene (responsible forthe production of a Tyr protein abnormallyprocessed with severely reduced enzymaticactivity). The mutant Tyrc-em locus encodes analtered Tyr protein that is incorrectly glycosylatedand partially retained in the ER, althoughconserving some detectable Tyr activity.

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FootnotesThis work was supported by funds from the Spanish Ministry of Science and Technology Bio2000-1653,Bio2003-08196 and from CAM 08.5/0046/2003 to LM, and SAF2003-03411 from the CICyT and FEDERto JCGB. The authors are most grateful to P. Glenister (MRC, Harwell, UK) for kindly rescueing Tyrc-em

and Tyrc-m mice from frozen embryos and for sharing unpublished initial description of the extremedilution mottled mutation; to V. Hearing for kindly providing αPEP7 and KDEL antibodies; to F. Solanofor useful comments; to A. Gutiérrez-Adán, S. Montalbán, and J. Fernández for helping in the rederivationof Tyrc-em and Tyrc-m mice to the CNB animal house, to M. Cantero and P. Cozar for technical assistancewith mice, and to G. Jeffery for revising the manuscript. Correspondence and request for materials shouldbe addressed to Lluís Montoliu (montoliu@cnb.uam.es).

1The abbreviations used are: Tyr and TYR, mouse and human tyrosinase protein, respectively; Tyr andTYR, mouse and human tyrosinase gene, respectively; L-DOPA, L-3,4-dihydroxyphenylalanine; Endo F,N-glycosidase F; Endo H, endoglycosidase H; ER, endoplasmic reticulum; LCR, locus control region.

2GenBank = GenBank Accession Number AY526904.

FIGURE LEGENDSFigure 1Phenotypic analysis of Tyrc-em mice. (A) From left to right, heterozygous extreme dilution mottled Tyrc-em

(Tyrc-em/Tyrc), homozygous extreme dilution mottled Tyrc-em and homozygous chinchilla mottled Tyrc-m

representative mice. All animals are in an albino outbred NMRI genetic background. Irises (B, E, H),whole-mounted retinae (C, F, I), peripheral retina at higher magnification (D, G, J) and histologicalanalysis of horizontal adult retinal sections (K, L, M) of heterozygous extreme dilution mottled Tyrc-em

(Tyrc-em / Tyrc) (B, C, D, K), homozygous extreme dilution mottled Tyrc-em (E, F, G, L) and homozygouschinchilla mottled Tyrc-m mice (H, I, J, M). Scale bars in B, E, H = 500 µm, in C, F, I = 100 µm and in D,G, J, K, L, M = 30 µm. Arrows in K, L, M depict variegation areas (i.e. RPE cells devoid of pigment).More choroid pigmentation can be see in M (above).

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Figure 2Measurement of Tyr enzymatic activity and melanin contents in whole-eye extracts from adultextreme dilution mottled mice (A) Tyrosine hydroxylase activity, measured by a radiometric assay usingtritiated tyrosine as substrate (n=5). (B) Melanogenic activity, estimated from the formation of aradioactive insoluble polymer using 14C-L-tyrosine as substrate (n=5). (C) Colorimetric measurement ofocular melanin contents (n=5). Bars represent the mean value (+/- SD) and correspond to albino NMRImice (Tyrc/Tyrc) (white bars), heterozygous extreme dilution mottled mutant mice (Tyrc-em/Tyrc) (light greybars), chinchilla mottled mutant mice (Tyrc-m/Tyrc) (grey bars), heterozygous YRT4 transgenic mice (34,42) (YRT4/- ; Tyrc/Tyrc) (dark grey bars) and heterozygous YRT2 transgenic mice (37) (YRT2/- ;Tyrc/Tyrc) (black bars). Statistically significant differences (paired t-Student tests) are indicated as follows:(*) p<0.05; (**) p<0.01; (***) p<0.001.

Figure 3Genomic analysis of Tyrc-em locus.(A) Schematic representation of Tyr+, Tyrc-em and Tyrc-m genomic loci. Restriction sites for Apa I (A),EcoR I (E) and Hind III (H) are indicated. White boxes correspond to DNA probes, black boxes to theLCR element while exons and reconstituted cDNA of the Tyr gene are depicted as grey boxes. A bentarrow indicates the transcription start site. Dashed lines show putative ends for the genomic translocationdescribed in Tyrc-m mutant mice (40). (B) High-resolution Southern blot from large size genomic DNAfrom wild-type (Tyr+), homozygous mutant extreme dilution mottled (Tyrc-em) and homozygous mutantchinchilla mottled (Tyrc-m) mice, digested with Apa I and EcoR I restriction enzymes and hybridised with aLCR-specific probe (probe D, 35). (C) Southern blot of mouse genomic DNA from the same threegenotypes indicated in (B) digested with Hind III and EcoR I restriction enzymes and hybridised with afull-length Tyr cDNA probe (pmcTyr1, 28).

Figure 4Evaluation of the glycosylation pattern and subcellular distribution of Tyr from the Tyrc-em mutantmice.(A) Extracts from HEK 293T cells transfected with the wild-type form or the indicated mutants clonedinto pcDNA3 (10 µg total protein per lane) were electrophoresed, blotted, and probed with αPEP7. Themembranes were then stripped and re-probed with an anti-ERK2 antibody (Santa Cruz), used as a loadingcontrol (lower blot). A representative blot out of three independent experiments is shown in (A), and theresults of quantitative analysis of the band intensities is displayed in (B) (expressed as % band intensityrelative to wild-type, mean ± s.e.m, n = 3).(C) Extracts from HEK 293T cells transfected with wild-type and extreme dilution mottled constructspcDNA3 (8 µg total protein per lane) were electrophoresed, blotted, and probed with αPEP7, with orwithout a previous deglycosylation treatment with EndoH or EndoF. The migration of molecularmolecular weight markers is shown on the left. C stands for undigested control extracts, and eH and eF forextracts digested with Endo H or Endo F, respectively.(D) Subcellular distribution of Tyr in the Tyrc-em mutant mice. Co-localisation analysis by confocalmicroscopy of wild-type Tyr+ and mutant Tyrc-em, Tyr∆6 (47) and TyrMeB (47) tyrosinase in HEK 293Ttransfected cells with an ER marker. αPEP7 (green) was used to identify the tyrosinase proteins andKDEL (red, 12) was used to detect ER. Co-localisation of antibodies is shown in orange-yellow colour.Nuclei were counterstained with TO-PRO3 (blue). Scale bar = 5 µm

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Table I

Residual enzymatic activity and kinetic constants (KM y Vmax) of Tyrc-em tyrosinasea

Protein Tyrosine hydroxylase activity KM (µM) Vmaxb Vmax/ KM

(µu/mg protein)

Tyr+ 2920 + 20 (100%) 79 + 10 2.11 + 0.07 0.027

Tyrc-em 368 + 13 (13%) 139 + 30 1.36 + 0.19 0.010

aValues are given as mean + SD for n=3 independent experiments.bVmax values are expressed in arbitrary units, as reported (47).

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Alfonso Lavado, Concepcion Olivares, Jose Carlos Garcia-Borron and Lluis Montoliucoding and non-coding genomic alterations

Molecular basis of the extreme dilution mottled mouse mutation: A combination of

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