Role of pH in determining the cell-type-specific residual activity of glucocerebrosidase in type 1 Gaucher disease.

Role of pH in determining the cell-type-specific

residual activity of glucocerebrosidase in type 1

Gaucher disease.

S van Weely, … , J M Tager, J M Aerts

J Clin Invest. 1993;

91(3)

:1167-1175.

https://doi.org/10.1172/JCI116276

.

The properties of control and 370Asn-->Ser glucocerebrosidase, the frequently encountered

mutated form of the enzyme in type 1 Gaucher disease, were studied in vitro as well as in

situ. The catalytic properties of purified 370Asn-->Ser glucocerebrosidase were highly

dependent on the assay conditions. The enzyme was deficient in activity towards substrate

and in reactivity with the irreversible inhibitor conduritol B-epoxide (CBE) when activated by

the bile salt taurocholate. In the presence of more physiological activators, the lysosomal

activator protein saposin C and phosphatidylserine, the 370Asn-->Ser enzyme was near

normal in kinetic properties at pH values approximately 5, but not at higher pH. In intact

fibroblasts, the enzymic activity of the 370Asn-->Ser glucocerebrosidase and its reactivity

with CBE were found to be clearly deficient. However, in intact lymphoblasts from the same

patients, the behavior of the mutant enzyme was near normal. The catalytic efficiency of

370Asn-->Ser glucocerebrosidase in situ was also found to be highly pH dependent. When

intact lymphoblasts were cultured in the presence of permeant weak bases, which increase

the pH of acidic intracellular compartments, the catalytic efficiency of the mutant enzyme, as

assessed by its reactivity with CBE, became markedly impaired. Our findings indicate that

the intralysosomal pH in the intact cell can be expected to have a critical influence on the

activation state of 370Asn-->Ser glucocerebrosidase and […]

Research Article

Role of pH

in

Determining the Cell-type-specific Residual Activity of

Glucocerebrosidase

in

Type

1

Gaucher Disease

S.vanWeely,*M.vandenBerg,*J. A.Barranger,t M.C. SaMiranda,§J. M.Tager,* andJ. M. F.G. Aerts*

*E. C. SlaterInstitutefor Biochemical Research, UniversityofAmsterdam, Academic MedicalCentre, 1105AZAmsterdam, The Netherlands;

*Department

ofHumanGenetics, University ofPittsburgh, Pittsburgh, Pennsylvania15261; and §Instituto Genetica Medica

JacintoMagalhaes, 4000 Porto,Portugal

Abstract

The

properties

of control and

370Asn

-- Ser

glucocerebrosidase,

thefrequently encountered mutated form ofthe enzyme in type 1 Gaucher disease,werestudied in vitroas well as insitu.

The

catalytic properties

of

purified 37Asn

-* Ser

glucocere-brosidasewerehighly dependentontheassayconditions. The

enzyme wasdeficientinactivitytowards substrateandin reac-tivity with the irreversible inhibitor conduritol B-epoxide

(CBE) when activated by the bile salt taurocholate.Inthe pres-enceofmorephysiological activators,thelysosomal activator

protein saposin

C and

phosphatidylserine,

the

37OAsn

-- Ser

enzyme was near normal in kinetic properties at pH values

5,but not athigher pH.

Inintactfibroblasts, the enzymic activity of the

370Asn

Serglucocerebrosidaseanditsreactivity with CBEwerefound

to beclearly deficient. However,in intactlymphoblasts from

the samepatients, the behavior ofthe mutant enzyme was near normal. Thecatalyticefficiency of

370Asn

-- _Ser

glucocerebro-sidaseinsituwasalsofoundtobehighly pH dependent.When

intactlymphoblastswerecultured inthepresenceofpermeant weakbases, which increase the pH of acidic intracellular com-partments, the

catalytic efficiency

ofthe mutant enzyme, as

assessed by its reactivity with CBE, became markedly

im-paired.

Our

findings

indicatethat theintralysosomal pHin the in-tact cell can be expected to have acritical influence on the activationstateof

37OAsn

-- Ser

glucocerebrosidase

andits

abil-ity to hydrolyse substrate. This phenomenon may partly

un-derly the marked heterogeneity in clinical manifestation of

Gaucher diseaseamongpatients with this mutated formof

glu-cocerebrosidase. (J. Clin. Invest. 1993. 91:1167-1175.) Key words:conduritolB-epoxide-glucosylceramide lipidosis *

lyso-somal storagedisorder*saposin C Introduction

Gaucher disease (glucosylceramidosis) is a recessively

in-herited

lysosomalstorage disorder in which the activity of

glu-cocerebrosidase (EC 3.2.1.45)is deficient. Theclinical

mani-Addresscorrespondence to J. M. F. G.Aerts, Publication Secretary, E.C.SlaterInstitute for BiochemicalResearch, University of

Amster-dam, Academic Medical Centre, Meibergdreef 15, 1105 AZ

Amster-dam, The Netherlands.

Receivedfor publication 26 May 1992 and in revisedform 4 July 1992.

festation

ofthe disease isvery

heterogeneous

with respecttoage

ofonset and

neurological

involvement. In

patients

with the mostfrequently encountered form,type1, the clinical

presenta-tion may varyfromcaseswithsevere

splenomegaly

andbone

deteriorationtocaseswithout clearclinicalcomplications

( 1).

Type2and type3Gaucher disease

patients develop

neurologi-cal complications in infancy or childhood, respectively ( 1). Gaucher disease is genetically heterogeneous. Several

muta-tionsinglucocerebrosidasehave been identified(2-6),and it

has becomeclear that patientsmayhave eithertwoidenticalor, moregenerally,twodifferentmutant

glucocerebrosidase

alleles (see e.g., references 7 and 8). Most type 1 Gaucher disease

patients

carry atleastonecopyofa mutant

glucocerebrosidase

allele in whichamutationat

position 1,226

in thecDNAleads

to

substitution

of G for A(cDNA

nucleotides

are numbered

starting

with theupstreamATGatnumber1

[9]).

Thisresults

inasubstitutionof serine for asparagineatamino acidposition 370 in thematureprotein (8, 10, 11). The

370Asn

-- Ser

gluco-cerebrosidase ispresentinnormalamounts( 12-15).In

vitro,

thisenzymeis only poorly active undermost

conditions;

how-ever,whenactivatedatacidic pH by

negatively

charged phos-pholipid and the natural lysosomal activator protein saposin C,

formerly

referredto as

sphingolipid

activator

protein

2

(for

a

review,seereference 16), its activity isnearnormal(17).

An-other

frequently

encountered form of mutated

glucocerebrosi-dase has a CforTsubstitution atnucleotide

position

1,448, resulting inaproline for leucine substitutionatposition444in

the matureprotein. This mutation has been detected in various phenotypes of Gaucher disease. Most

patients

whoare

homo-zygousfor this mutation developaneuronopathiccourseofthe disease (7, 11, 18-20). The

4"Leu

-* Pro

glucocerebrosidase

results in reduced enzyme stability(15-21). In athird

com-monlyencountered allele,aG insertion in the

glucocerebrosi-dase gene at cDNA

position

84results in

production

ofa

trun-catedprotein (22).

Theconsequencesofthe

370Asn

-o_Sersubstitution for

cata-lytic efficiency of glucocerebrosidase in situ are notyet fully understood.Ithas

previously

beenobserved

by

usthat thestate

of activation of

370Asn

Ser

glucocerebrosidase,

and

con-comitantly

its

catalytic efficiency,

differs in homogenates of fibroblasts and lymphoblasts obtained from the same type 1

Gaucherdisease

patients (23).

Wehave nowstudiedthe prop-erties ofglucocerebrosidase inintact fibroblasts and

lympho-blasts.

First,

thedegradation ofafluorescent analogue of gluco-cerebroside by intactcellswasmeasured.Second,the

reactivity

of

glucocerebrosidase

withtheirreversible inhibitor conduritol B-epoxide

(CBE)'

wasassessed.Thisinhibitor isanalogousin 1.Abbreviations usedinthis paper: CBE,conduritol B-epoxide;

C6-NBD glucosylceramide, 6-[N-7-nitrobenz-2-oxa-1,3

diazol-4-yl-ami-nocaproyl ]sphingosyl

3-D-glucoside;

4-MU-,B-glucoside,

4-methylum-belliferyl-fl-D-glucoside.

J.Clin. Invest.

©TheAmerican Society forClinical Investigation, Inc.

002 1-9738/93/03/1167/09 $2.00

structureto the transition state of gluconolactone formed dur-ing hydrolysis of glucocerebroside. In contrast to the natural transition statecompound, the epoxide is abletoforma cova-lent bond with anasparticacid group in the catalytic site, thus irreversibly inhibiting the enzyme (2, 24). It has elegantly been shown by Bieberich and Legler (25) that CBE rapidly enters lysosomes in intact cells and effectively inhibits theenzymein situ.

Our investigation revealed differences between properties ofglucocerebrosidase in intact fibroblasts and in intact lympho-blasts from type 1Gaucher disease patients with 370Asn-- Ser

glucocerebrosidase. The possible role of the natural activator

protein saposin C and lysosomal pH in this connection was investigated. The results are reported here, and the

implica-tions arediscussed.

Methods

Materials. CBE waspurchased from Biomol Research Laboratories

(Philadelphia,PA), sodium taurocholategradeAwaspurchasedfrom Calbiochem(SanDiego, CA);andphosphatidylserine,and

4-methyl-umbelliferyl-#-D-glucoside

(4-MU-3-glucoside) was purchased from

SigmaImmunochemicals(St. Louis, MO).C6-NBDglucosylceramide

(6-[N-7-nitrobenz-2-oxa- 1,3diazol-4-yl-aminocaproyl] sphingosyl

f-n-glucoside)was akindgiftfrom Dr. G. Schwarzmann(University of

Bonn, Bonn,Germany).Allotherchemicalswereof the purestgrade

commerciallyavailable.

Cell linesofGaucher disease patients.Propertiesof

glucocerebrosi-dasewerestudied in cultured skin fibroblasts and EBV transformed B lymphocytes(lymphoblasts)from Gaucher diseasepatientswitha de-finedglucocerebrosidasegenotype; i.e. genotypes 370Asn Ser/370Asn

Ser,"4Leu -* Pro/44Leu-- Proand370Asn -*

Ser/4"Leu

-* Pro. Fibroblastswereobtained byskinbiopsiesandlymphoblastswere ob-tainedby transformationofperipheralbloodBlymphocyteswithEBV

(23, 26). The cellswerecultured in RPMI 1640containing 10%(by

volume) FCS and bicarbonate with 5% CO2 in the gas phase. The

glucocerebrosidasegenotype of thepatientswasdeterminedby

allele-specific oligonucleotide hybridization (26). In thecase ofpatientC (Table I),it hasnot asyet been excluded that the mutated 44Leu-* Proallele containsadditional mutations(alsoreferredto as

complex

patternalleles).The cellbiologicalconsequencesof the 44Leu-0 Pro

mutationandoneof thecomplexpattern allelesareknowntobe simi-lar; i.e. theproductionofarapidly degradedglucocerebrosidase ( 15).

TheGaucher diseasepatients homozygousorheterozygousfor the

370Asn-- _{Ser allele variedmarkedlyin}severityofnonneuronopathic manifestation of thedisorder.Thepatientshomozygousfor the 44Leu

-- _{Pro allele had}developedneurological complications alreadyin

in-fancy (type 2)oronlyinchildhood(type3).

Consistent with earlierfindings, itwas noted usingPAGE in the presence of sodium dodecyl sulphate followed by Western

blotting

(27), that theamount ofglucocerebrosidase antigen was normal in fibroblasts ofpatients homozygousfor the370Asn-- Ser allele(i.e.,_type 1patients)and verystrongly reduced in fibroblasts ofpatients homozy-gous for the 44Leu-- _{Pro allele(i.e.,type2 and3}patients).Incells frompatientswho weregenetic compounds carrying both alleles, inter-mediateamountsofcross-reactiveglucocerebrosidaseweredetected.

Preparation ofantisera. A monospecific rabbit antiserum to

hu-manplacental glucocerebrosidasewasprepared as described (28). A

monospecificrabbitantiserumtosaposinC waspreparedasdescribed

(29).

Immunoaffinity chromatographic purification of

glucocerebrosi-dase. Glucocerebrosidasewaspurifiedfrom various materials by immu-noaffinity chromatography with immobilizedantiglucocerebrosidase

monoclonalantibodies 8E4 and 2C7asimmunosorbens exactlyas

de-scribedpreviously(30).

TableI. RelativeSpecificActivityof Glucocerebrosidase

Immunopurified

from Cultured Skin Fibroblasts of Gaucher

Patients

Relativespecific activity measured in the presenceof

Genotype Patient Taurocholate/Triton PS/saposin C

370/370 A 16, 14 77,88 B 15,8 79, 84 370/444 C 21, 20 92, 87 D 22 89 444/444 E 81,73 86,82 F 85 81 Control (n=4) 88-115 92-107

Identicalamountsof cross-reactive glucocerebrosidase in preparations from cultured skin fibroblasts of control subjects and Gaucher disease patientswerebound inamicrotiter plate in which monoclonal anti-humanglucocerebrosidaseantibody 8E4wasimmobilised, as de-scribed in Methods. The relativespecific activityis the enzymic

ac-tivityperamountofcross-reactiveglucocerebrosidaseina prepara-tion fromacell lineofaGaucherdiseasepatientaspercentage of the mean value forpreparationsfrom four different control fibroblast cell lines. Theactivity ofboundglucocerebrosidasewasdetermined with two different assay mixtures, each well containing

150,pl

assay mixture. The reactionwasstopped by addition of 100pl 1 M

gly-cine-NaOH, pH 10.6. Thetaurocholate/Triton assaymixture con-tained 5 mM

_{4-MU-0-glucoside,}

0.1%(by volume)TritonX-100,

0.5%(mass/vol)sodium taurocholate, 100:200 mMcitric

acid/so-diumphosphate, pH 5.0;andthephosphatidylserine/saposinC assay mixture contained 5mM4-MU-/3-glucoside,

10,qg/ml

phosphatidyl-serine,2.4

_,g/ml

saposinCpreparation,100:200mMcitric

acid/so-diumphosphate,pH5.0. Thespecific activityof control

glucocere-brosidase immobilizedtomonoclonalantibody8E4was1.8 and 1.4 nmol/h per mgproteinwhen measured inthe presence of

taurocho-late/TritonandPS/saposin C, respectively. 370,370Asn - Serallele;

444,

44Leu

-. Pro allele.

Purification ofsaposin C. The activatorprotein waspurified

ac-cordingtotheproceduredescribed(31).

Assessmentofactivatorproteinlevel.The levelof activator protein wasdetermined byafunctional test as described (12).

The assayof glucocerebrosidase with

4-MU-13-glucoside

as

sub-strate.Fibroblastswereharvestedbytrypsinisation and lymphoblasts bycentrifugation.The cellswerewashed inisotonic PBS and homoge-nizedasindicated in thetext.Thecontribution of lysosomal

glucocere-brosidasetothe total,B-glucosidase activitywasdeterminedusingCBE. Enzyme preparations were simultaneously preincubated for 5 min without CBE,orwith 5mMCBE to ensure that allglucocerebrosidase was inactivated by the irreversible inhibitor, and subsequently ,B-gluco-sidaseactivitywasmeasured inthe absence and presence of 1 mMCBE under theconditions indicated in thetext.TheCBE-inhibitable

3-glu-cosidaseactivityis caused by the lysosomalglucocerebrosidase(31).

Proteinwasdeterminedasdescribed by Lowryetal.(32) with BSA as standard.

Measurementofrelativespecificactivityofglucocerebrosidase. Pre-vious studies have shown that monoclonalanti-(human

glucocerebro-sidase) antibody8E4 has thesamebinding affinityfor control,370Asn

-- Sersubstituted and 44Leu -- Pro substitutedglucocerebrosidase (12, 13, 15). Thus, identicalamountsof various types ofglucocerebro-sidase willbeboundto aspecificamountof immobilized monoclonal

consistsofamixture oftwoforms ofglucocerebrosidase, the bound

glucocerebrosidasewill beacomparable mixture. Thesebinding char-acteristics of the monoclonal antibody8E4 allowasimple analysisof thespecificactivityof mutatedglucocerebrosidaseperamountof en-zymeprotein as described previously ( 12). Briefly, identical amounts ofantiglucocerebrosidase monoclonal antibody 8E4 wereimmobilized

towellsof a microtiter plate. The wells were incubated with enzyme preparations containing an excess of glucocerebrosidase antigen. The

activity ofboundglucocerebrosidase was measuredwiththe fluoro-genic4-MU-3-glucosideassubstrate as described in the legendofTable I. Fluorescence in the wells of themicrotiter plate was determined (Fluoroscan; FlowLaboratories Inc., McLean, VA) with excitation at 366nmandemissionat450 nm.

Measurementof hydrolysis of C6-NBD glucosylceramide in intact cells.C6-NBDglucosylceramide was complexed with BSA as described (13). Cells were incubated for 24 h at 370C with 5

_juM

lipid-BSA complex in the absence or presenceof excess CBE. The cells were har-vested, andlipids were extracted and separated by thin layer chromatog-raphy. Thefluorescentlipids were scrapedoff,extracted, and quanti-fiedfluorimetricallyasdescribed ( 13).

Immuno-electron microscopy. Cultured human skin fibroblastsand

EBV-lymphoblastswerefixed and preparedforimmunogold labeling andelectronmicroscopyfollowingthe proceduredescribed for

fibro-blasts(33). Frozen cellsamples were cryosectioned and

immunola-beled, andexamined usingatransmissionelectronmicroscope (model 300;Phillips Electronic Instruments Co., Mahwah, NJ) at an accelerat-ing voltageof 60kV.

Results

Enzymic properties

ofpurified glucocerebrosidase

Glucocerebrosidase waspurified byimmunoaffinity

chroma-tography from cultured skin fibroblasts. The recovery of en-zyme inmonomeric form (31 )wasalways> 70%onthe basis ofenzymic activityasmeasured in the presenceof

taurocho-late.

Relative

specific activity. The enzymic activity per

amount

ofcross-reactiveglucocerebrosidase relatedtothat ofcontrol enzyme(i.e., the relative specific activity) wasdeterminedfor enzyme preparations from fibroblasts from various types of Gaucherdisease patients(seeTable I). When measured inthe presenceof taurocholateandTritonX-100,therelativespecific

activity of

glucocerebrosidase

in preparations of cells of pa-tients

_homozygous

for the 370Asn -- Ser allele is

abnormally

low, andthatofenzymeinpreparations fromcellsofpatients

homozygousforthe"4Leu-> Pro alleleiscloseto normal. The

relative specific activity of glucocerebrosidase inpreparations

from cells of patients who are genetic compounds for the

370Asn -- Ser and"4Leu Proallelesis also low,

_suggesting

thattheycontain predominantly

370Asn

-- Ser

glucocerebrosi-dase molecules.

Whenbound glucocerebrosidase is measured in the pres-ence of

phosphatidylserine

and activator protein the relative

specific

activity

ofenzymein the preparations fromfibroblasts

ofall thepatients isnear normal (see TableI).In other words, under these

conditions,

the"70Asn-* Ser

glucocerebrosidase

is

normalized.

The amountof cross-reactive glucocerebrosidasecan be

es-timated by comparingthe datafor relativespecific activity with valuesfor specific activityinthe corresponding cell extracts. In

thisway,itwas calculated that theconcentrationof

glucocere-brosidase antigen is 80-100% infibroblastsfrom patients with genotype 370Asn -.

_Ser/370Asn

-* _Ser, < 10% in cells from

patients

with genotype "4Leu --

Pro/444Leu

-- Pro and - 30% in cells from

patients

carryingboth alleles.

Inactivation

by

CBE. The

irreversible

inhibition of

gluco-cerebrosidase by CBE was determined for enzyme purified from fibroblasts ofindividuals with different

glucocerebrosi-dase genotypes.Fig. 1 A shows theresults whenenzyme

prepa-rationswereincubatedwithinhibitorin thepresenceof

tauro-cholate and Triton X-100, and enzyme activity was

subse-quently determined under the same conditions. The

glucocerebrosidase purified

fromfibroblasts of thepatient

ho-mozygousfor the "4Leu-*Proallelewasinactivated ina man-nersimilartocontrolenzyme,but the inactivation ofenzyme

purifiedfrom cells ofthepatienthomozygousfor the 370Asn-*

Serallelewas very slow. Theinactivationofenzymepurified from cells ofthegenetic compound proceededwith interme-diatekinetics. When theenzymepreparationswereincubated with inhibitor in thepresenceofphosphatidylserineand

activa-torproteinatpH 5.0, and theenzymeactivitywasdetermined under thesameconditionadifferent picturewasobtained(see

Fig. 1 B). Under these conditions, the behavior of

glucocere-brosidase purified from fibroblasts of all the Gaucherdisease

patientswas notclearly different from that of controlenzyme.

Thissuggeststhat in the presence of phospholipid and activator

protein

the370Asn -* Ser

glucocerebrosidase

isnot

only

'nor-malized' in activity towards substrate, but also in reactivity withCBE.

Analogous findingstothe results presented in Table I and Fig. 1 were made forglucocerebrosidase purified from urine samples, spleens, and lymphoblasts from type I Gaucher dis-easepatients (notshown).No significant differenceswere

ob100 - 0-0f -0 A PRE-INCUBATION (MIN) B 0 0 Go 0 60 PRE-INCUBATION (MIN) Figure 1. Inactivation ofpurifiedglucocerebrosidase byconduritol

B-epoxide.Glucocerebrosidase waspurifiedfrom fibroblasts of indi-viduals with knownglucocerebrosidasegenotypeby immunoaffinity chromatographyasdescribedpreviously (30).Thepurifiedenzyme waspreincubatedat roomtemperature for the times indicated with 25

,M

CBE in 100:200 mM citricacid/sodium phosphatebuffer,pH 5.0,containing0.1%(mass/vol)BSA and10% (byvolume) ethylene

glycol. Thepreincubationmixturefurthermore contained either 0.1% (byvolume) Triton X-l00 and 0.5%(mass/vol)sodium taurocholate (A)or10,g/ml phosphatidylserineand 2.4

.g/ml

saposinC prepa-ration (B).Subsequently,the enzymepreparationwasdiluted10-fold

inpreincubation mixture from which CBEwasomittedandtowhich 5 mM

4-MU-f3-glucoside

wasadded. Theactivityisexpressedasa

percentage of that found withoutpreincubation.Theresults ofa typ-icalexperimentareshown.Similarresults wereobtained in threeto

five otherexperimentsandwithdifferent batches of CBE. The values in parentheses indicate thespecific activityofimmunopurified

gluco-cerebrosidase(nanomolesper hour permilligramofprotein)in the presence oftaurocholate/Triton X-100. o, Control(2.8);., 370Asn

served between enzymepreparationsfrom the various sources

examined.

It is of importance to note that thereactivity of

glucocere-brosidase with CBE istosomeextent agoodreflectionofthe activity of the enzyme towards substrate. This was also ob-served when membrane suspensions were tested instead of

pure enzymepreparations.

Enzymic properties ofglucocerebrosidase in intact

fibroblasts

and

lymphoblasts

Activity

towards substrate.

The glucocerebrosidase activity in cultured fibroblasts and lymphoblasts from type 1 Gaucher diseasepatients and control subjectswasdeterminedby load-ing cells with C6-NBD glucosylceramideasdescribedin Meth-ods. Recentlyweestablished the existence of anonlysosomal glucocerebrosidase activity incells thatis insensitive toCBE.

Theproperties of the nonlysosomal glucocerebrosidase, which is not deficient in Gaucher disease materials, are to be de-scribed inaseparatepublication. Toallow discrimination

be-tweenthelysosomal

(CBE-sensitive)

andnonlysosomal (CBE-insensitive)

glucocerebrosidase

cellswerepretreated with and withoutan excess of CBE. Thelysosomal

glucocerebrosidase

activitywasdeterminedas the CBE-inhibitable hydrolysis of C6-NBD

glucosylceramide

(Table II). The residual

activity

in fibroblasts of

patients

with the370Asn -* Serallelewas

always

lessthan that in lymphoblasts from thesame

patients.

Inactivation by CBE. The kinetics ofthe

irreversible

inhibi-tion

of glucocerebrosidase

uponincubation of intact cells with CBE was examined

(Fig.

2). Glucocerebrosidase in control

fibroblasts

and in"4Leu -- Pro/"4Leu_{-> Pro fibroblasts}was

clearly inactivatedmore

rapidly

thantheenzyme in370Asn->

Ser/370Asn

->Serfibroblasts whenenzymeactivitywas

subse-quently

measured either in thepresence of taurocholateand Triton X-100

(Fig.

2A)orin thepresenceof

phosphatidylser-ine and activator

protein

at acid pH

(Fig.

2 B). In370Asn -o

Ser/44Leu-> Profibroblasts,the inactivation of

glucocerebro-sidase

appeared

tobemore

rapid

whenthe

activity

was subse-quently measured in thepresenceof taurocholate and Triton TableII. GlucocerebrosidaseActivityIn Situ towardsC6-NBD

Glucosylceramide inFibroblasts andLymphoblasts from a ControlSubject andaTypeI GaucherDisease Patient

PercentconversionofC6-NBD glucosylceramide

toceramide+sphingomyelinin

Fibroblasts Lymphoblasts

CBE-

CBE-Cells from -CBE +CBE Inhibitable -CBE +CBE Inhibitable

Controlsubject 60 21 39 26 13 13

Gaucherpatient 35 29 6 21 12 9

Cells grown in RPMI 1640 culture medium with 5%CO2in the gas phasewerepreincubatedwithorwithout 100_,MCBE for24 h at

37°C. Next,C6-NBD glucosylceramide/BSA(5

MM:5

,uM)wasadded. After 24 hat37°C, the cells were extensively washed and the NBD-lipidswereextracted and analysed using thin layer chromatog-raphyasdescribed in Methods. Glucocerebrosidase activity is ex-pressedasthe percent conversionofC6-NBD glucosylceramideto

C6-NBD ceramideandC6-NBD sphingomyelin. Fluorescencein other lipidswasalways <4%. Results of one experiment are shown; similar resultswereobtained intwoindependent experiments.

100 -U 0-0 A

L

60 0 PRE-INCUBATION (MIN) 60 PRE-INCUBATION (MIN) Figure 2. Inactivation of glucocerebrosidase in intact fibroblasts by conduritol B-epoxide. Cells werepreincubatedin culture medium for

different periodsof timewith 100 ,uM CBE. Afterharvestingand washing with PBS, the fibroblasts were homogenized in 50 mM po-tassiumphosphate,pH6.5, containing0.25%(by volume)Triton

X-100.

f-Glucosidase

activity was determined with 5 mM

4-MU-f-glucosideas substrate either in 100:200 mM citric acid/sodium phosphate, pH 5.2,0.1% (by volume) Triton X-100 and 0.2% (mass/vol) sodium taurocholate (A) or in 100:200 mM citric

acid/sodium phosphate,pH 5.0, 10

_Ag/ml

phosphatidylserine, and 2.4

Ag/ml

saposin C preparation (B). The results of a typical experi-mentareindicated. Similar data were obtained in at least two

inde-pendent experiments.

o,

Control;

.,

370Asn

--

Ser/370Asn

-- _Ser;A,

170Asn

Ser/44Leu

-- _Pro;A,

44Leu

--

Pro/4"Leu

-* Pro.

X-

100

(Fig. 2A) thanwhen itwasmeasuredwith phosphati-dylserineandactivatorprotein (Fig. 2 B). This apparent dis-crepancyresults from the fact that with activatorprotein and

phosphatidylserine,

one measures predominantly

370Asn

--Serenzyme,which is slowly inactivated, whereas in the pres-enceof taurocholateandTritonX-

100,

44Leu->Pro enzyme

contributes

significantly

tothe totalactivity.

Comparison ofdiferent

cell

types. Cultured

fibroblasts

and

lymphoblastswereincubated with CBE for different periods of time, and subsequently, enzymic activity ofglucocerebrosidase

was measured in the presence of taurocholate and Triton

X-l00.

TableIIIshows

again

thatlysosomal glucocerebrosidase in

intact fibroblasts from

patients

with the370Asn-- Ser allelewas

clearly less sensitivetoinhibition by CBE comparedto enzyme

in

corresponding

cellsofacontrol subjectand apatient homo-zygous for the

44Leu

-> Pro

glucocerebrosidase

allele. How-ever,

glucocerebrosidase

in cultured lymphoblasts froma

con-trol

subject,

a

patient

homozygous for the codon 370

substitu-tion,

and a

patient

with both the codon 370 and codon 444

substitutionswas comparably inactivated by CBE. This

cell-type-specific

difference between fibroblasts and lymphoblasts

wasalso observed in threetype1_{Gaucher disease patients with}

one allelewith the codon 370mutationand an as yet unidenti-fiedmutated allele(not shown).

Immunocytochemical analysis ofglucocerebrosidase and

activator

protein saposin

C

in

cultured

cells

The apparentdifference in

glucocerebrosidase

activity in intact lymphoblastsandfibroblasts could,inprinciple,becausedby a

relatively higher concentration of activator protein in lyso-somes oflymphoblasts. To testthispossibility,the relative

in-tralysosomal concentrations of

glucocerebrosidase

and

activa-tor protein in fibroblasts and lymphoblasts were studied by

immunoelectron microscopy using specific antisera. Fig. 3 showssomeexamples of double-labeling of

glucocerebrosidase

TableIII. Inactivation of GlucocerebrosidaseinIntact Cellsby Conduritol B-Epoxide

Activity nmol/h per

mgprotein in CBE-inhibitable activity (percent nonpreincubated ofthat innonpreincubated cells) cells afterpreincubation with CBE for

Cell type -CBE +CBE 10 20 40 60

min Fibroblasts Control 384.2 1.0 80 66 45 30 370/370 37.8 0.7 95 87 79 68 370/444 20.7 0.7 90 81 64 52 444/444 17.2 0.4 82 69 48 32 Lymphoblasts Control 17.1 0.4 75 55 29 16 370/370 1.5 0.3 80 63 32 21 370/444 1.2 0.3 78 59 33 17

Cells grown in RPMI 1640 culture mediumcontaining10%(by vol-ume) FCS with 5%CO2in the gasphasewereincubatedat370C

with 100

_AM

CBEfor different periodsoftime, washed extensivelyat 40Cwith PBSto remove excessinhibitorandharvested. Next, the cellswerehomogenizedin 50 mMpotassium phosphate, pH 6.5, containing0.25%(by volume) TritonX-100.f-Glucosidaseactivity

wasmeasuredwith 5 mM

4-MU-3-glucoside

assubstrate in100:200

mMcitric acid/sodium phosphate (pH5.2) containing0.1% (by vol-ume) TritonX-100and 0.2%(mass/vol)sodium taurocholate. At eachpreincubationtimepoint,enzymeactivitywasmeasuredin the presence of 1 mM CBE inonesample and in the absenceofCBE in

aparallel sample. Enzymeactivitywasrelatedtototal cellularprotein.

TheCBE-inhibitable enzymic activityateachtimepointis expressed

aspercentage of that in cellsnotpreincubatedwithinhibitor. The

re-sultsofatypical experimentareshown. Analogous resultswere

obtained inatleast threeindependent experiments. 370,

"0Asn

-Serallele;444,44Leu-- _Proallele.

a control subject anda

patient

with the genotype370Asn

-Ser/44Leu

Pro.

Quantification

ofthe

differently

sized

gold

particles

in lysosomes shows that the ratio of labeled activator proteintolabeled

glucocerebrosidase

isnotsignificantly

differ-entin

fibroblasts

andlymphoblasts of the control

subject

and theGaucherdisease

patient (Table IV).

The amount

ofactiva-tor

protein

wasalso

quantified

bymeansofafunctionaltest in

whichtheability

of

denaturedprotein preparations from

differ-entcelltypes toactivate

purified

glucocerebrosidase

was

mea-sured. Asshown in Table V,the amountof activator

protein

was greaterinfibroblaststhaninlymphoblasts, but there was nodifference between control and Gaucher fibroblasts,or

be-tweencontrol andGaucherlymphoblasts. Thus, theapparent

difference in

properties

of

glucocerebrosidase

in lymphoblasts

andfibroblastsfrom

patients

with

370Asn

Ser

glucocerebro-sidasecannotsimplybeattributedto ahigheramountof intra-lysosomal activator protein in the lysosomes ofthe former

cells.

Effect ofpH

on

activity

of3'0Asn

-* Ser

glucocerebrosidase

Pureenzyme. Theinfluence ofpH onenzymicactivity of

puri-fied

splenic

370Asn

-> Serglucocerebrosidase and control en-zyme wasstudiedusing acetate andcitricacid/sodium phos-phatebuffersin the pH range 4.0-6.0. Fig. 4 A shows that the

activity

of

370Asn

-* Ser

glucocerebrosidase

in the presenceof

activator protein andphospholipidhasaverysharp pH

opti-mum. At pH values > 5.4 in acetate buffer and > 5.2 in citric

acid/sodium phosphatebuffer, the mutated enzyme is mark-edly reduced in specific activity. It was noted that in the pres-ence ofhigher amounts of activator protein, the specific

activ-ity of370Asn-- Serglucocerebrosidasewasalso closetonormal

atpHvalues between 4.3 and5.0, but not at pH values > 5.0 (not shown;see also

[17]).

It appears that the 370Asn -) Ser

substitutioninglucocerebrosidaseleads to an increasedpH

de-pendency ofthe enzyme with respecttoactivationbyactivator

protein. Particularlyathigher pH values,the mutated enzyme

is poorly activated by saposin. Fig. 4 B shows that also the

reactivity

of

370Asn

-* Ser

glucocerebrosidase

with CBE is ex-traordinarily pH dependentinthe presence of activatorprotein andphospholipid.Athigher pH values,the mutated enzyme is

dramatically lesssensitive for CBE-induced inactivation

com-paredtocontrolenzyme.

Similar

resultstothosepresented in

Fig.

4were obtained withenzymeisolatedfrom fibroblastsandlymphoblasts from

patients

homozygous

forthe

370Asn

Serallele.

Furthermore,

370Asn-- Serglucocerebrosidase isolated from 3T3fibroblasts

transfected with site-directed mutagenized human glucocere-brosidasecDNA (15) showed acomparable pH dependency (not shown).

Manipulation ofintralysosomalpH with weak bases.

Intact fibroblasts andlymphoblastswere exposedtoNH4Cl, aweak base, toincrease the

intralysosomal

pH, asdescribed by Oh-kuma and Poole (34). These authors have shown that in

mouse macrophagesaddition of increasing concentrations of NH4Cl leadsto aconcomitant increasein pHof intracellular acidiccompartments(includinglysosomes), asmonitored by

the extent of quenching ofthe fluorescence ofendocytosed

dextran-fluorescein

isothiocyanate. For instance, onaddition of 10 mM

NH4Cl,

the pH of such compartmentsincreases from - 4.8 to - 6.2. Fig. 5 shows that with increasing

amountsof

NH4Cl,

glucocerebrosidase in cells becomes less

sensitive

toinactivation by CBE,aswould be expectedfromthe

results of Fig. 4 B. This effect of NH4Cl is reversible (not

shown).Itshould be noted that inthe absenceofNH4Cl gluco-cerebrosidase is muchmoreeffectively inactivatedby CBE in

Gaucher disease

lymphoblasts

thanin Gaucher disease fibro-blasts. However, at increasing concentrations of NH4C1, the rate of inactivation of glucocerebrosidase in lymphoblasts

re-semblesthat infibroblasts fromthe same Gaucherdisease

pa-tient. Similar observationswere madeusing methylamine(not

shown).

The findings presented in Figs. 4 and 5 suggest that the

intralysosomal pH to which glucocerebrosidase is exposed

mighton the average belower in cultured lymphoblasts than in

fibroblasts.As anapproximatemeasure ofthe pH inlysosomes

(and other acidic compartments),we measured the accumula-tion of

radioactively

labeledweak base chloroquine (35). We observed that about seven times more radioactively labeled

chloroquinewastrapped bylymphoblaststhan by fibroblasts when related to the amount of glucocerebrosidase present in the two cell types,whereasthe immunogold labeling studies suggest that the concentrationof glucocerebrosidase is about

twiceashighin lysosomes of fibroblasts than of lymphoblasts

(seeTable IV). Thissuggests that the average intralysosomal pH ishigherinfibroblaststhan in lymphoblasts.

Assessmentofactivation state ofglucocerebrosidase. When

pro->:<:

Ace

4

Table IV. Quantification

of

GoldLabeling ofGlucocerebrosidase andSaposinCinLvsosomal Structures in Fibroblasts

and Lymphoblasts fromaControlSubjectand a TypeI Gaucher Patient

Number ofgold particles/lysosome in fibroblasts Number ofgold particles/lysosome inlymphoblasts

Cell line Antigen Mean±SEM n Range Mean±SEM n Range

Control 3-Glu 8.1±1.2 44 0-34 3.9±0.5 83 0-31 SaposinC 11.6±2.1 43 0-51 5.4±0.7 84 0-30 SaposinC/f-Glu 1.61±0.28 38 0-9 2.00±0.37 69 0-18 Gaucher f-Glu 6.0±0.9 20 0-15 2.5±0.7 33 0-16 SaposinC 9.1±1.4 20 1-22 5.8±1.0 33 0-29 SaposinC/3-Glu 2.04±0.43 19 0-7 2.98±1.10 25 0-29

Forexperimentaldetails,seethelegendtoFig.3.Lysosomalstructurespresent in electronmicrographsofatypical immunolabeling experiment

werequantified. The numberoflysosomalstructuresanalysed is indicated in the table.

3-Glu,

glucocerebrosidase.

portion

of

glucocerebrosidase

remains in activated

form,

as

reflected bya high ratio of enzymic activitymeasured in the

absenceof additional activators

compared

tothat measuredin

thepresenceof taurocholate (see

[23]).

The bile salt abolishes anyoriginal activationofglucocerebrosidaseand activatesfully

all enzymes present(23). Table VIshows that the

activity

in

the absenceof activator is

relatively higher

ina

homogenate

of lymphoblasts than that inahomogenate of

fibroblasts.

When

the cellsarepretreated with NH4C1toincreasethepHof

intra-cellularacidiccompartments (seeabove), this difference

dis-appearsand

glucocerebrosidase

is only poorly activeinthe ab-sence of additional activator in homogenates from both cell

types. This

finding

supportsthe

assumption

thaton the aver-age, glucocerebrosidase ispresentina moreactivatedstatein intact lymphoblasts than in

fibroblasts

because ofan

intralyso-somal pH difference.

Some ofourfindings are consistent with those of earlier

studies.

First,

it has been

reported by

Grabowski and

co-workers(36, 37) that the mutantform ofglucocerebrosidase in Ashkenazi Jewishtype 1 Gaucher diseasepatientsreferredto as

group B mutant enzymeisless

efficiently

inactivated

by

CBE

thancontrolenzyme either in fibroblastextracts orinpartially purified splenic preparations. It is now almost certain that group B mutant enzymeisidenticaltothe370Asn-- Ser

gluco-cerebrosidase(2). Second,it has beenreportedthat glucocere-brosidaseinintact fibroblasts from Ashkenazi Jewish Gaucher

diseasetype 1 patientswaslessefficientlyinactivatedbyCBE

compared toenzyme in cells from control

subjects

and from otherGaucher diseasepatients (36). Thus,inthe studies cited

above,

particular

stress waslaiduponthe ethnic

background

of type 1Gaucher disease

patients.

Wehavefoundthat therateof

inactivation of

glucocerebrosidase

by CBE in fibroblasts from

Discussion

The

properties

of the370Asn-- _Ser

_{glucocerebrosidase}

_{in vitro}

are

intriguing:

Undermostassayconditions, this mutated en-zymeisimpaired in activitytowards substrate. However,inthe presenceofthe naturalactivator protein saposinC and

phos-phatidylserine,

theenzymefunctions ina nearnormal

fashion

at an

appropriate acidic

pH. To

obtain

information

on the properties ofthis mutatedenzymeinsitu,theirreversible inhib-itor CBE has been used, anapproach thatwasdevelopedand

used

previously

by other

investigators

(25, 36, 37). Although it is obvious that the reaction of glucocerebrosidase with CBE involves onlypartofthetotalreaction mechanism ofsubstrate

hydrolysis (discussed inmoredetail in [2

]),

analysis of

CBE-induced inactivation of glucocerebrosidase isinformative be-causethereactivity of (mutant) glucocerebrosidase withCBE appears to

reflect

to alargeextenttheability ofenzyme to be

active towards substrate.

TableV.LevelQfActivator Protein(Saposin C) in Fibroblasts andLymphoblasts fromaControlSubject

anda Type 1 Gaucher Patient

Cell type Cellline Activator

ULing protein

Fibroblasts Control 0.58

Type 1 Gaucher 0.53

Lymphoblasts Control 0.21

Type 1 Gaucher 0.26

Cellextracts wereboiledfor 5 min, centrifuged, and the supernatants were testedfor their capacity to stimulate the enzymic activity of

purifiedplacental glucocerebrosidase asdescribed(12). 1 U of activa-tion isdefinedasthe amountof activator protein that stimulates the

activityofglucocerebrosidaseby afactorof100.

Figure 3. Immunocytochemicalvisualisation of glucocerebrosidase and saposin C. Fibroblasts and lymphoblasts of a control subject and a Gaucherdisease type 1 patient heterozygous for the370Asn-> Ser and 44Leu -*Pro glucocerebrosidase substitutions were washed in PBS, fixed,

cryosectionedandprocessedfor immunogold double labeling as described (33). The immunoglobulins were visualized with protein A conjugated with 10-nm gold particles (antiglucocerebrosidase) and with protein A conjugated with 5-nm gold particles (anti-saposin C). Identical results were obtained when glucocerebrosidase was visualized with a 5-nm gold probe and the activator protein was visualized with a 10-nm gold probe. (A) Fibroblasts of the controlsubject;(B) fibroblasts of the Gaucher patient; (C) lymphoblasts of the control subject; and (D) lymphoblasts of

3- 2- 0-A 100-I - 0-4 5 pH 6 B 4 5 6 pH Figure 4. Influence of pHonthe properties of 370Asn Ser

gluco-cerebrosidase, purified from spleen. The effect ofpHontheproperties

ofimmunoaffinity purified control and370Asn-- Ser

glucocerebrosi-dase inthepresenceof10,g/mlphosphatidylserineand2.4

;g/ml

saposin Cwasdetermined.(A) The activity of glucocerebrosidasewas

determined with 5 mM4-MU-,B-glucosideassubstrate inthepresence

of 50mMacetatebuffer(o, * )or50:100 mM citric acid/sodium

phosphate buffer (A, A) varying in pH from 4.0to6.0.oandA, con-trol; and A,370Asn-- Ser/370Asn-- Ser.(B)Theinactivation of glucocerebrosidase by CBEwasdetermined in 50:100mMcitric

acid/sodium phosphate buffer (pHrange4.0-6.0). Enzyme activity wasdeterminedasdescribed in the legend of Fig. 1.o,Control; .,

370Asn-. Ser/370Asn Ser.

370Asn Ser/ 370Asn -*Serpatients and,toalesser extent, in

fibroblasts from themorefrequentlyencounteredpatients

het-erozygousforthismutation,is slow.However,we werenotable todistinguishtype 1Gaucherdiseasepatientswith Ashkenazi

Jewish backgroundfrompatientswith non-Jewishbackground

onthebasisofCBE-inducedinactivation.

During this study, the important observation was made

thatthe activation state ofglucocerebrosidasein thelysosomes as measured in situ differs in fibroblasts and lymphoblasts.

This finding substantiates earlierstudies carried outwith ho-mogenates fromthetwo celltypes(23).Thecell-type-specific

difference isparticularly evidentin the caseof370Asn -- Ser

100

-o

0-Table VI. Activation State of Glucocerebrosidase inHomogenates ofControl Cells

Glucocerebrosidaseactivity

inhomogenate with

Cellspreincubated Noadditions Taurocholate/Triton

Cell type for30 minwith (A) X-100 (B) A/B

nmol/h

Fibroblasts Noadditions 79.6 153.2 0.52

25 mM NH4C1 57.2 159.1 0.36

Lymphoblasts Noadditions 10.0 14.1 0.71

25 mMNH4Cl 5.3 13.9 0.38

Cells wereharvested,washed in PBS andhomogenizedin 10:20 mM

citric acid/sodium phosphate buffer,pH 4.0, bybrief sonication.

Enzyme activity was measured in two reaction mixtures. The reaction mixturewithoutan addedactivator contained100:200 mM citric

acid/sodium phosphate,pH 4.0, and 5 mM4-MU-,B-glucoside.The other reaction mixturecontained 100:200 mMcitricacid/sodium

phosphate, pH 5.2,0.1%(by volume)TritonX-100 and 0.2% (mass/

vol)sodiumtaurocholateas activators, and 5 mM

4-MU-f3-glucoside.

Assays wereperformedin the absence andpresence of1 mM CBE todeterminethecontribution ofglucocerebrosidasetothetotal

(3-glucosidase activity.The results of atypical experimentaregiven. Similarresults wereobtainedin two otherindependent experiments.

glucocerebrosidase,

the catalytic properties ofwhich are so

strongly

determined

by thestateof

activation

ofthe enzyme.

Our

studies

suggestthatthe intralysosomalpHdetermines thestate

of activation of glucocerebrosidase.

Again, this is

par-ticularly

relevant for370Asn-*Ser

glucocerebrosidase:

the

acti-vation of thismutant enzyme by activator protein is extraordi-narilypHsensitive. Thus,acell-type-specific differencein

in-tralysosomal

pH could

explain

thedifference in properties of

370Asn

- _Ser

_{glucocerebrosidase}

_{in intact fibroblasts}and

lym-phoblasts. Moreover,variability between individuals in intra-lysosomal

conditions

mayunderlie the heterogeneity in

sever-ity of

the

clinical expression of

Gaucher disease in individuals with thesame

glucocerebrosidase

genotype.Inthis connection, it

is of

interest to mention that some homozygotes for the

370Asn

->Ser

glucocerebrosidase mutation

are not oronlyvery

mildly

affected clinically

(8, 38), and thatastrict correlation between

glucocerebrosidase

genotype and Gaucher disease clinicalphenotype has notalways been noted; e.g., see

refer-ence39.InGaucher

disease, glucocerebroside accumulation

is restrictedto

macrophage-like cells,

Itwill, therefore, be

ofinter-est to

establish

the

intralysosomal

pH and thestate

of

activa-tion of

glucocerebrosidase

in macrophages

from

patients

with differentdegreesofseverity oftheclinicalmanifestation.Such

studiesareunderway.

50 0

mMNH4CI mM NH4CI

Figure5. Influence ofammoniaoninactivation ofglucocerebrosidase

in intact cellsby conduritol B-epoxide.Cellswerepreincubatedfor 30minat37°Cinculture mediumcontainingdifferent

concentra-tions ofNH4Cl. Next, 100 uM CBEwasadded to the medium and

the cellswereharvestedimmediatelyor 1hlater.Glucocerebrosidase

activitypermilligram total cellular proteinwasdeterminedas

de-scribedinthelegendtoTable III. The results ofatypical experiment

areshown.Similar resultswereobtainedin atleasttwoindependent experiments. (A) Lymphoblasts; (B) fibroblasts. o, Control; *,

"70Asn-- _Ser/370Asn-- _Ser;

A, "70Asn--

Ser/4"Leu

-- Pro.

We are most grateful to Ms. Betty Brouwer-Kelder, Marinellavan

Leeuwen,and AnnekeStrijland for their excellent technical assistance,

andtoDr. M.Horowitz and Dr. E. M. Petersen forprovidinguswith

fibroblastandEBV-lymphoblastcelllines.Furthermore,wewouldlike

tothank Dr. G. Schwarzmann for hisgenerousgiftofC6-NBD

gluco-sylceramide.Wethank Ms.WendyvanNoppenand ElsbethVlugt-van

Daalen for theirhelpinthepreparation ofthemanuscript.

Thisworkwassupported bygenerousgrantsfromThe National

GaucherFoundation,U.S.A.(grantnumberNGF 17),and the Junta

0

Nacional de Investigacao CientificaeTechnologica, Portugal (grant number 87388). J. M. F. G. Aertsis anEstablishedInvestigatorofthe

Royal Netherlands Academy of Sciences.

References

1. Barranger, J. A., and E. I. Ginns. 1989. Glucosylceramide lipidosis: Gaucher disease. In The Metabolic Basis of Inherited Disease. C. R.Scriver,A. L. Beaudet, W. S. Sly, andD.Valle, editors. McGraw-Hill Inc., NY. 1677-1698.

2.Grabowski, G. A., S. Gatt, andM.Horowitz. 1990. Acid,3-glucosidase: enzymologyand molecularbiologyof Gaucher disease. Crit. Rev. Biochem. Mol. Biol.25:385-414.

3. Hong, C. M., T.Ohashi,X. J.Yu, S.Weiler,and J.A.Barranger. 1990. Sequence oftwoallelesresponsiblefor Gaucher disease. DNA Cell Biol. 9:233-241.

4.Latham, T.,B.D.M.Theophilus,G.A.Grabowski,and F. I. Smith. 1991. Heterogeneity ofmutationsin theacidbeta-glucosidasegene of Gaucher disease

patients.DNACell Biol. 10: 15-21.

5. Beutler, E., T.Gelbart,W.Kuhl,A.Zimran, and C. West. 1992. Mutations inJewishpatientswith Gaucherdisease. Blood. 79:1662-1666.

6.Beutler, E. 1992. Gaucher disease: New molecularapproachestodiagnosis andtreatment.Science(Wash. DC).256:794-799.

7.Tsuji, S.,P. V.Choudary, B. M.Martin,B. K.Stubblefield,J.A.Mayor, J.A.Barranger, and E. I. Ginns. 1987.Amutation in the human

glucocerebrosi-dase gene inneuronopathicGaucher's disease.N.Engl.J. Med. 316:570-575. 8.Zimran,A.,E.Gross, C. West, J.Sorge,M.Kubitz,and E.Beutler. 1989. Prediction ofseverityofGaucher's diseaseby identificationof mutationsatDNA level. Lancet.8659:349-352.

9.Sorge, J., C. West, B. Westwood, and E. Beutler. 1985. Molecularcloning

andnucleotide sequenceofthe humanglucocerebrosidasegene. Proc. Natl. Acad. Sci. USA.82:7289-7293.

10. Tsuji,S., B. M.Martin,J.A.Barranger, B. K.Stubblefield, M. E. La-Marca, and E. I. Ginns. 1988.Geneticheterogeneityin type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazicindividuals.Proc.Natl. Acad.Sci. USA.85:2349-2352.

11. Theophilus, B., T. Latham, G.A. Grabowski,and F. I. Smith. 1989. Gaucherdisease: molecularheterogeneityandphenotype-genotypecorrelations. Am.J.Hum.Genet. 45:212-225.

12. Aerts, J.M. F.G., W. E.Donker-Koopman,S.Brul,S.vanWeely,M.C. SaMiranda,J.A.Barranger,J. M.Tager, andA.W.Schram. 1990.Comparative

studyonglucocerebrosidasein spleens frompatientswith Gaucher disease.

Bio-chem. J. 269:93-100.

13. Van Weely, S.,M. B. vanLeeuwen, I. D. C. Jansen,M. A.C. de Bruyn,

E.M.Brouwer-Kelder,A.W.Schram, M. C. SaMiranda,J.A.Barranger,E. M.

Petersen, J. Goldblatt,etal. 1991. Clinical phenotypeofGaucherdiseasein relationtopropertiesofmutantglucocerebrosidasein culturedfibroblasts. Bio-chim.Biophys.Acta. 1096:301-311.

14.Grace,M. E.,P. N.Graves,F. I.Smith,andG.A.Grabowski. 1990. Analyses ofcatalytic activityandinhibitorbinding ofhuman acidfl-glucosidase

by site-directedmutagenesis.Identificationofresidues criticaltocatalysisand evidence forcausalityoftwoAshkenazi Jewish Gaucher disease type 1mutations. J.Biol. Chem. 265:6827-6835.

15.Ohashi,T., C.M.Hong, S.Weiler, J. Tomich, J. Aerts,J.Tager, andJ.

Barranger. 1991. Characterization of human glucocerebrosidase from different mutantalleles.J.Biol. Chem. 266:3661-3667.

16.O'Brien, J. S., and Y. Kishimoto. 1991. Saposin proteins: structure,

func-tion,and rolein humanlysosomalstorage disorders. FASEB (Fed. Am. Soc. Exp.

Biol.) J.5:301-308.

17. Aerts, J. M. F. G., M. C. Sa Miranda, E. Brouwer-Kelder, S. van Weely, J.A.Barranger, and J. M. Tager. 1990. Conditions affecting the activity ofgluco-cerebrosidase purified from spleens of control subjects and patients with typeI

Gaucherdisease.Biochim.Biophys. Acta. 1041:55-63.

18. Masuno, M., S. Tomatsu, K. Sukegawa, and T. Orii. 1990. Non-existence ofatight associationbetween a44leucineto proline mutation andphenotypes of Gaucherdisease: high frequency of aNciIpolymorphism in the

nonneurono-pathicform. Hum. Genet. 84:203-206.

19.Dahl, N., M. Lagerstrom, A. Erikson, and U. Pettersson. 1990. Gaucher disease typeIII(Norrbottnian type)is caused by a single mutation in exon 10 of

theglucocerebrosidase gene. Am.J. Hum.Genet. 47:275-278.

20.Glew,R.H., V. Gopalan, C. A. Hubbell, E. Beutler, J. D. Geil, and R. E. Lee. 1991. A case ofnonneurologic Gaucher's disease that biochemically resem-bles the neurologic types. J. Neuropathol. & Exp. Neurol. 50:108-117.

21.Grace, M. E., A. Berg, G. He, L. Goldberg, M. Horowitz, and G. A. Grabowski. 1991.Gaucher disease: Heterologous expression of two alleles

asso-ciated withneuronopathic phenotypes. Am. J. Hum. Genet. 49:646-655. 22.Beutler, E., T.Gelbart, W. Kuhl, J. Sorge, and C. West. 1991. Identifica-tion of the secondcommon Jewish Gaucher disease mutation makes possible

population-based screeningfor the heterozygous state. Proc. Natl. Acad. Sci. USA. 88:10544-10547.

23. SaMiranda, M. C.,J. M. F.G. Aerts,R. Pinto, S. vanWeely,J.A. Barranger, and J. M. Tager. 1990.Activity of glucocerebrosidase in extracts of different cell types from type 1 Gaucher diseasepatients.Clin. Genet. 38:218-227.

24.Legler, G. 1988.Beta-glucocerebrosidase: mechanistic studies with cova-lent and non-covacova-lentinhibitors. In Lipid Storage Disorders. Biological and Bio-medical Aspects. R. Salvayre,L. Douste-Blazy, and S. Gatt,editors. Plenum Press, NY. 63-72.

25. Bieberich, E., andG. Legler. 1989. Intracellularactivityoflysosomal

glucosylceramidase measured with 4-nonylumbelliferyl (3-glucoside. Biol. Chem.

Hoppe-Seyler.370:809-817.

26.Firon, N.,N.Eyal, E. H.Kolodny,and M. Horowitz. 1990. Genotype

assignmentin Gaucherdisease by selectiveamplificationof theactive glucocere-brosidase gene. Am. J. Hum. Genet. 46:527-532.

27. Aerts, J. M. F.G.,W. E.Donker-Koopman,M. K.vanderVliet,L. M. V. Jonsson, E. I.Ginns,G. J. Murray, J.A.Barranger, J.M.Tager, andA.W. Schram. 1985. Theoccurrenceoftwoimmunologically distinguishable /3-gluco-cerebrosidases in human spleen. Eur. J. Biochem. 150:565-574.

28.Ginns,E.I., R. 0.Brady, S.Pirruccello,C. Moore, S.Sorrell,F.S.Furbish,

G. J.Murray, J. Tager, and J. A. Barranger. 1982. Mutations of glucocerebrosi-dase:discrimination ofneurologicandnon-neurologic phenotypesof Gaucher disease. Proc.Natl. Acad. Sci. USA. 79:5607-56 10.

29.Fujibayashi,S., and D.A.Wenger. 1985.Synthesisandprocessingof

sphingolipid activatorprotein-2 (SAP-2) in cultured human skin fibroblasts. Clin. Chim. Acta. 146:147-156.

30. Aerts, J. M. F.G.,W. E.Donker-Koopman,G. J. Murray, J.A.Barranger,

J.M. Tager, andA.W.Schram. 1986.Aprocedurefor therapid purification in

high yieldof human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies.Anal. Biochem. 154:655-663.

31. Aerts, J. M. F. G., W.E.Donker-Koopman, C. vanLaar,S. Brul, G. J. Murray, D.A.Wenger, J.A.Barranger, J. M. Tager, and A. W.Schram. 1987.

Relationshipbetween thetwoimmunologicallydistinguishable forms of gluco-cerebrosidase intissue extracts. Eur.J.Biochem. 163:583-589.

32. Lowry, 0. H., N. J. Rosebrough,A.L. Farr, andR.J. Randall. 1951. Proteinmeasurementwith the Folin phenol reagent. J.Biol. Chem. 193:265-275.

33.Wiemer,E. A.C., S. Brul, W. H. Just, R. van Driel, E. Brouwer-Kelder, M.

vanden Berg, P. J.Weijers, R. B. H. Schutgens, H. van den Bosch, A. W. Schram,

etal. 1989. Presenceofperoxisomalmembraneproteinsinliver andfibroblasts

frompatientswith the Zellweger syndrome and related disorders:evidence forthe existence ofperoxisomal ghosts.Eur. J.Cell Biol.50:407-417.

34.Ohkuma, S.,and B. Poole. 1978. Fluorescence probemeasurementofthe

intralysosomalpH inlivingcells and theperturbation of pH byvariousagents. Proc.Natl. Acad.Sci. USA. 75:3327-3331.

35. Hollemans, M., R. P. J. Oude Elferink, P. G. de Groot, A. Strijland, and

J.M. Tager. 1981. Accumulationofweakbases inrelation tointralysosomal pH in cultured human skin fibroblasts. Biochim. Biophys. Acta. 643:140-151.

36.Grabowski,G. A., T.Dinur,K.M.Osiecki, J. R. Kruse, G. Legler, and S. Gatt.1985. Gaucher disease types 1, 2, and 3: differential mutations of the acid

0-glucosidaseactive site identified with conduritol B epoxidederivatives and

sphingosine.Am.J. Hum.Genet.37:499-510.

37.Grabowski,G. A., K.Osiecki-Newman,T.Dinur, D. Fabbro, G. Legler, S.

Gatt,andR.J.Desnick. 1986. Human acid ,B-glucosidase: use of conduritol B

epoxidederivativestoinvestigatethecatalytically active normal and Gaucher disease enzymes. J. Biol.Chem. 261:8263-8269.

38.Zimran,A., T.Gelbart,B.Westwood, G.A.Grabowski, and E. Beutler. 1991.Highfrequencyofthe Gaucherdiseasemutation atnucleotide 1226 among AshkenaziJews. Am.J Hum. Genet.49:855-859.

39.Sidransky,E., S.Tsuji,B.M.Martin, B. Stubblefield,andE. I. Ginns. 1992.DNAmutation analysis of Gaucher patients. Am.J.Med. Genet.