American Journal of Medical Genetics 116A:238–242 (2003)

Biochemical Characterization of Two (C300F, P425T)Arylsulfatase A Missense Mutations

Ana Marcao,1 Heidi Simonis,2 Frank Schestag,2 M. Clara Sa Miranda,1 and Volkmar Gieselmann2*1Instituto de Biologia Molecular e Celular, University of Porto, Porto, Portugal2Institut fur Physiologische Chemie, Rheinische Friedrich Wilhelms Universitat, Bonn, Germany

Metachromatic leukodystrophy (OMIM250100) is a lysosomal storage disease causedby the deficiency of arylsulfatase A (ARSA,EC 3.1.6.8). This disease affects mainly thenervous system, because patients cannotdegrade3-O-sulfo-galactosylceramide(sulfa-tide), a major myelin lipid. Here we describethe characterization of the biochemicaleffects of two arylsulfatase A missense muta-tions, P425T and C300F. Transfection ex-periments demonstrate the expression ofresidual ARSA enzyme activity for P425T,but not for C300F substituted ARSA. Relativespecific activity determination showed thatthe P425T substituted enzyme has retainedabout 12% of specific enzyme activity,whereas the C300F substituted enzyme isreduced to less than 1%. Pulse-chase experi-ments reveal that both mutant proteins areunstable, with a half life of less than 6 hr.Increased secretion upon addition of NH4Clindicates that the mutant proteins can passthe Golgi apparatus and thus are not de-graded in the endoplasmic reticulum (ER),but in the lysosomes. This is supported byexperiments, which demonstrate the pre-sence of mannose-6-phosphate residues ontheoligosaccharidesidechainsof themutantproteins. Addition of the cysteine proteaseinhibitor leupeptin increases the amount ofARSA activity in cells expressing the P425Tsubstituted enzyme, whereas no increase inactivity was seen with C300F substitutedARSA. � 2002 Wiley-Liss, Inc.

KEY WORDS: arylsulfatase A; metachro-matic leukodystrophy; lyso-somal storage disease

INTRODUCTION

Arylsulfatase A is the lysosomal enzyme that cata-lyzes the first step in the degradation of sulfatide(3-O-sulfogalactosylceramide), one of the major sphin-golipids in myelin. Deficiency of this enzyme causesmetachromatic leukodystrophy, adisease that is charac-terized by sulfatide accumulation in different tissues.Oligodendrocytes and Schwann cells are the mostaffected. Thus, patients develop a progressive demyeli-nation and a wide variety of neurological symptoms.metachromatic leukodystrophy (MLD) is clinicallyheterogeneous, presenting with three different formsclassified according to the age of onset: late infantile,juvenile, and adult forms [for review see Von Figuraet al., 2001].

The ARSA locus is located on chromosome 22q13; thegene is divided into eight exons encompassing 3.2 Kb(GenBank X52150). It codes for an enzyme of 507 aminoacids [Stein et al., 1989; Kreysing et al., 1990]. A largenumber of mutations has been reported in the ARSAgene. Most are private and only a small number ofthem were biochemically characterized [Von Figuraet al., 2001].

A genotype–phenotype correlation exists in MLD:patients homozygous for alleles associated with noresidual activity usually present the late infantile formof the disease, while patients with at least one allele ortwo alleles coding for an enzyme with some residualactivity present less severe, juvenile or adult forms.This correlation was first suggested by Polten [Poltenet al., 1991] and has been supported by biochemical datafrom several reports [Bohne et al., 1991; Fluharty et al.,1991; Leinekugel et al., 1992; Kappler et al., 1994;Hess et al., 1996; Coulter-Mackie et al., 1997; Hermannet al., 2000].

In this article, two defective ARSA proteins arebiochemically characterized. Activity, stability, andsorting of these enzymes are analyzed and the obtaineddata are discussed according to the clinical phenotype ofthe patients.

Grant sponsor: FCT (Portugal); Grant number: PRAXIS XXI/BD/16058/98.

*Correspondence to: Prof. Volkmar Gieselmann, Institut furPhysiologische Chemie, Nussallee 11, 53115 Bonn, F.R.G.E-mail: gieselmann@institut.physiochem.uni-bonn.de

Received 5 November 2001; Accepted 7 June 2002

DOI 10.1002/ajmg.a.10822

� 2002 Wiley-Liss, Inc.

MATERIALS AND METHODS

Patients

Themutations characterized in this study were foundin two Portuguese patients. One of these patientspresented the late infantile clinical form of MLD andwashomozygous for thealteration that changes cysteine300 into phenylalanine. The other patient presented thejuvenile form of the disease and was a compoundheterozygote for mutation P426L [Polten et al., 1991]and amutation that changes proline 425 into threonine.Both of these patientswere previously reported [Marcaoet al., 1999].

Material

Taq polymerase, LIPOFECT AMINE Reagent, G418,and cell culture media were from Life Technologies(Eggenstein, Germany). Oligonucleotides were pur-chased from MWG-Biotech (Ebersberg, Germany).Restriction enzymes and DNAmodifying enzymes werefrom New England Biolabs (Frankfurt am Main,Germany) or Life Technologies. Endoglycosidase Hwas from Roche. [35S]-methionine (specific activity>600Ci/mmol) and [32P]-orthophosphate (specific activ-ity >3,000 Ci/mmol) were from Amersham Phar-macia (Freiburg, Germany). PANSORBIN was fromCalbiochem-Novabiochem (Schwalbach, Germany).Other reagents were from Sigma or Merck.

In Vitro Mutagenesis

MutationsC300F andP425Twere introduced into thepAlter mutagenesis vector from Promega according tothe protocol supplied by themanufacturer. The insert ofthe vector contained an SV40 early promoter, the ARSAcDNA and a polyadenylation sequence from the pBEHARSA vector described previously [Artelt et al., 1988;Stein et al., 1989]. The oligonucleotides that were usedto introduce mutations have the following sequences:C300F-50TGCTCCGGACTCTTGCGGTTTGGAAAGGG-A30 and P425T-50ACTGCTCATGAGACCCCGCTCC-TCTATGAC30. The introduction of these mutationswas confirmed by DNA sequencing.

Activity, Stability and IntracellularLocalization of Mutant ARSAs

BHK-21 cells were transiently transfected with themutagenesis vector using lipofectamine, as described[Hermann et al., 2000]. After 48 hr, the cells wereharvested and ARSA activity was measured in cellularextracts using the artificial substrate p-nitrocatechol-sulphate. Total protein determination andWestern blotanalysis were also done.

The mutated ARSA cDNAs were subcloned into theexpression vector pBEH [Artelt et al., 1988]. Aneomycincassette, containing a neomycin resistance gene with anSV40 promoter and a polyadenylation signal was insert-ed at the unique Aat II restriction site. To generatestably transfected cell lines, Ltk� cells were transfected,using lipofectamine, and selected for two weeks with1mg/ml G418. After this time, metabolic labeling,

immunoprecipitation, and digestion of ARSA withendoglycosidase H were done, as described before[Gieselmann et al., 1992], with minor modifications.AnARSAanti-serum, raisedagainst recombinantARSAin rabbits, was used.

RESULTS

ARSA Activity in Transiently TransfectedBHK-21 Cells

The mutations causing the P425T and C300F aminoacid substitutions have recently been described, buttheir deleterious effect on ARSA activity expression hasnot been confirmed. To verify that the mutations arepathologic, amino acid substitutions P425T and C300Fwere introduced into the normal ARSA cDNA by in vitromutagenesis. The mutated cDNAs were transientlyexpressed in BHK-21 cells.

Five independent transient transfections were donefor each mutation.

Figure 1 shows that enzyme activity expression isseverely reduced for mutant P425T and completelyabsent for mutant C300F. To investigate ARSA relativespecific activities (Table I), we determined the enzymeactivity and the amount of ARSA cross-reacting mate-rial via Western blot analysis in the same cell lysates.The results reveal that the P425T substituted proteinhas considerable residual specific activity of about 12%of normal, whereas the specific activity of the C300Fsubstituted ARSA is less than 1%.

It has previously been demonstrated that somemutant ARSA proteins can be stabilized in culturedcells by the addition of the protease inhibitor leupeptin[Von Figura et al., 1983]. Upon addition of leupeptin the

Fig. 1. ARSA activity in transiently transfected BHK-21 cells. ARSAactivity was calculated as the average of five independent transfectionexperiments and is expressed as mU ARSA/mg total protein. Bars indicatestandard deviation. To determine endogenous activity of BHK-21 cells, thesame vector, but with an insert encoding a nonrelated protein, was alsotransfected and ARSA activity was measured. The bottom shows the resultof aWestern blot analysis of cell homogenates of transfected cells confirmingthe presence of ARSA cross-reacting material.

Arylsulfatase A Missense Mutations 239

ARSA activity in the cells increases and the catabolismof sulfatide is normalized. To investigate whether theP425T and C300F substituted enzymes can also bestabilized by cysteine protease inhibitors, leupeptinwasadded to transiently transfected cells and enzymeactivity was determined. Table II shows that additionof leupeptin leads to a considerable increase of enzymeactivity in the cells expressing the P425T substitutedenzyme. However, the protease inhibitor has no influ-ence on ARSA activity in cells expressing the C300Fsubstituted ARSA.

Stability and Intracellular Localization of theMutant ARSA Polypeptides

To investigate the effects of the amino acid substitu-tions on the stability of themutant proteins, pulse chaseexperiments were performed.

Stably transfected Ltk� cells expressing wild-typeARSA,C300F, andP425TsubstitutedARSAwerepulse-labeled for 3 hr with [35S]-methionine and chased up to72 hr.

Figure 2 shows that the wild-type enzyme is stableduring the entire chase period, whereas both mutantenzymes are rapidly degraded. More than half of themutated proteins are already degraded after 6-hr chase,an almost complete degradation occurring after 24 hr.

Premature degradation of mutant ARSA can eitheroccur in the endoplasmic reticulum (ER) or in thelysosomes. To investigatewhether themutated proteinscan leave the ER, pulse-chase experiments in thepresence of NH4Cl were done. NH4Cl interferes withthe cellular sorting of newly synthesized lysosomal

enzymes in a post Golgi compartment, targeting themimproperly to the secretory pathway. If upon addition ofNH4Cl mutant proteins can be found in the media ofcultured cells, this indicates that these enzymes musthave passed the Golgi apparatus and thus are able toleave the ER [Chang et al., 1988]. Cells were pulselabeledwith [35S]-methionine and chased in the absenceor presence of NH4Cl. ARSA was immunoprecipitatedfrom cells and media. The amount of C300F and P425TsubstitutedARSAsecreted into themedium increases inthe presence of ammonium chloride (Fig. 3) whichindicates that the mutant proteins are not arrested inthe ER.

The synthesis of themannose6-phosphate residues onthe oligosaccharide side chains of lysosomal enzymesoccurs in the Golgi apparatus. Thus, demonstration ofthe presence of phosphate residues on N-linked oligo-saccharide side chains is another proof forGolgi passageof a lysosomal enzyme. Ltk� cells expressing themutated proteins and the wild-type ARSA were pulse-labeled for 5 hr with [32P]-orthophosphate. Afterimmunoprecipitation the proteins were digested withendoglycosaminidase H, which removes high mannose-type oligosaccharide side chains.

Figure 4 shows that bothmutant enzymes are labeledby [32P]. The radioactive label can be removed byEndoHdigestion, indicating its presence on high mannose typeN-linked oligosaccharide side chains.

DISCUSSION

We biochemically characterized two missense muta-tions in the ARSA gene. The mutations have been

Fig. 2. Stability of wild-type and mutated ARSA polypeptides. Stabletransfected Ltk� cells expressing wild-type or C300F and P425T substitutedARSA were metabolically labeled for 3 hr with [35S]-methionine and chasedfor the times indicated. ARSA was immunoprecipitated from the cell lysatesand subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresisand fluorography.

TABLE I. ARSA Relative Specific Activity*

WT P425T C300F BHK-21

ARSA activity–ARSA endogenous activity (mU) 301.5 39 3 0Western blot arbitrary units 2,427 2,654 4,216 0Relative specific activity 100% 12% 0.6% —

*To estimate the relative specific activities of the mutant enzymes, transiently transfected BHK-21 cells wereanalyzed for ARSA activity and ARSA cross-reactingmaterial, determined byWestern blot analysis. Western blotsignalswere quantified by laser scan densitometry of the X-ray films and are presented in arbitrary units. Relativespecific activity was calculated as the quotient between ARSA activity and the Western blot signal.

TABLE II. Effect of Leupeptin in the ARSA Activity ofTransiently Transfected BHK-21 Cells

ARSA activity (mU/mg protein

Leupeptin (100 mM)

Expressed protein � þLAP 11.40 8.90ARSA-C300F 7.77 10.64ARSA-P425T 11.89 45.85ARSA-WT 47.55 46.35

LAP, lysosomal acid phosphatase. BHK-21 cells were transiently trans-fected to express the wild-type or substituted ARSA indicated in the table.Control cells were transfected with a plasmid coding for lysosomal acidphosphatase (LAP) Leupeptin (100 mM) was added after the transfection.The cells were harvested and ARSA activity and protein were measured incell homogenates, 48 hr, after transfection.

240 Marcao et al.

reported previously but their biochemical consequenceshave not been examined so far. Here we show that about10% residual enzyme activity can be expressed from theP425T allele but not from the C300F allele. This is inaccordance with the finding that the P425T ARSA hasconsiderable residual specific enzyme activity, whereasthat of the C300F is at the limit of detection (Fig. 1,Table I).

Biosynthesis studies show that both mutant enzymesare more rapidly degraded than the wild-type ARSA.The latter is stable over a chase period of 72 hr, whereasthe mutant enzymes are partially and completely de-graded after 6hr and 24hr of chase, respectively (Fig. 2).

Decreased stability of mutant ARSA frequently con-tributes to the enzyme deficiency in MLD [Kafert et al.,1995; Hess et al., 1996]. Previous studies have shownthat the degradation of the mutant enzymes occurseither in the ER [Kafert et al., 1995; Hess et al., 1996] orin the lysosomes [VonFigura et al., 1983;Kreysing et al.,1993]. This means that the structural alterationsresulting from mutations in this protein, lead eitherto endoplasmic reticulum retention and degradation

[Kafert et al., 1995; Hess et al., 1996] or cause an in-creased sensitivity for lysosomal proteases [Von Figuraet al., 1983; Kreysing et al., 1993]. Here we demonstratean increased secretion upon addition of NH4Cl of bothmutant enzymes (Fig. 3). Since this drug interferes withlysosomal enzyme sorting in a post Golgi compartment,this suggests that these enzymes are not arrested inthe ER. This is supported by the presence of mannose6-phosphate residues on the oligosaccharide side chainsof the mutant enzymes (Fig. 4), because these residuesare generated in a post ER compartment.

The biochemical properties of the P425T mutationresemble those of a previously described amino acidsubstitution, which occurs in the neighboring aminoacid, P426L. The latter is one of the most frequentmutations found amongwhites [Polten et al., 1991]. TheP426L substituted enzyme is correctly synthesized andsorted, but is rapidly degraded in the lysosomes. Thisdegradation could also be partially blocked by leupeptin[Von Figura et al., 1983]. At neutral pHARSA is a dimerthat octamerizes in apHdependentmanner in the acidicenvironment of the lysosome. Octamerization of theP426L substituted enzyme is impaired and this allowscathepsin L to cleave the mutant enzyme [von Bulow,2002]. It seems likely that the P425T substitutiondescribed here, may also interfere with octamerizationexplaining its cysteine protease sensitivity. Thus, thismutation has biochemical properties, which are similarto those of the P425T mutation described here. Prolines425 and 426 are part of a PPLL motive that is stronglyconserved among different sulfatases [Von Figura et al.,2001]. Thus, it seems that independent of themutations,alterations of this motif have similar consequences,namely, increased intralysosomal susceptibility forcysteine proteinases.

The biochemical data presented here fit well into theestablished genotype–phenotype correlation of MLD.The juvenile patient carrying the frequent P426Lmutation in one allele was found to be a compoundheterozygote forP425Tmutation inARSA’s exon8.Bothmutations still allow for the expression of low residualenzyme activities and thus, explain the late-onsetphenotype of the patient.

The C300F allele was found in homozygosity in apatientwith the severe late infantile form of the disease.Wehave shownthatnoenzymeactivity canbeexpressedfrom the allele carrying this mutation. This enzyme isalso sorted correctly to the lysosomes and thus resem-bles the P425T ARSA. Since it also displays a compar-able stability in pulse-chase experiments, the mainreason for the difference in enzyme activity must be thereduced specific activity of the enzyme. Therefore, theaddition of leupeptin cannot increase the ARSA activityin cells expressing this mutant, because in contrast tothe P425T substituted enzyme, C300F is enzymaticallyinactive (Fig. 1, Table I).

ACKNOWLEDGMENTS

A.M. was supported by grant PRAXIS XXI/BD/16058/98 from FCT (Portugal). V.G. was supported by a grantfrom the DFG.

Fig. 3. Effect of NH4Cl on sorting of wild-type and mutant ARSApolypeptides. Ltk� cells stably expressing the wild-type or the mutantproteins were pulse-labeled for 2 hr with [35S]-methionine and chased for 12hr in the presence or in the absence of 10 mM NH4Cl. After immunopre-cipitation of ARSA from the medium and the cells, ARSA was analyzed bysodium dodecyl sulfate–polyacrylamide gel electrophoresis and fluorogra-phy.Thebars indicate thepercentage ofARSAcross-reactingmaterial foundin the cells (C) or in themedia (M) and show the average of two independentexperiments. 1, no chase; 2, 12-hr chase; 3–12-hr chase in the presence ofNH4Cl. For each protein, 100% ARSA¼C1þM1.

Fig. 4. Phosphorylation of mutated ARSA polypeptides. Ltk� cells,expressing wild-type or substituted ARSA polypeptides, were metabolicallylabeled for 5 hr with [32P]-orthophosphate. ARSA was immunoprecipitatedfrom the cell lysates and digested with endoglycosidase H before sulfate–polyacrylamide gel electrophoresis analysis.

Arylsulfatase A Missense Mutations 241

REFERENCES

Artelt P, Morelle C, Ausmeier M, Fitzek M, Hauser H. 1988. Vectors forefficient expression in mammalian fibroblastoid, myeloid and lymphoidcells via transfection or infection. Gene 68:213–219.

Bohne W, von Figura K, Gieselmann V. 1991. An 11-bp deletion in thearylsulfatase A gene of a patient with late infantile metachromaticleukodystrophy. Hum Genet 87:155–158.

Chang PL, Ameen M, Yu CZ, Kelly BM. 1988. Effect of ammonium chlorideon subcellular distribution of lysosomal enzymes in human fibroblasts.Exp Cell Res 176:258–267.

Coulter-Mackie MB, Gagnier L, Beis MJ, Applegarth DA, Cole DE, GordonK, Ludman MD. 1997. Metachromatic leukodystrophy in three familiesfrom Nova Scotia, Canada: a recurring mutation in the arylsulfatase Agene. J Med Genet 34:493–498.

Fluharty AL, Fluharty CB, Bohne W, von Figura K, Gieselmann V. 1991.Two new arylsulfatase A (ARSA) mutations in a juvenile metachro-matic leukodystrophy (MLD) patient. Am J Hum Genet 49:1340–1350.

Gieselmann V, Schmidt B, Von Figura K. 1992. In vitro mutagenesis ofpotential N-glycosylation sites of arylsulfatase A. J Biol Chem 267:13262–13266.

Hermann S, Schestag F, Polten A, Kafert S, Penzien J, Zlotogora J,Baumann N, Gieselmann V. 2000. Characterization of four Arylsulfa-tase A missense mutations G86D, Y201C, D255H, and E212Dcausing metachromatic leukodystrophy. Am J Med Genet 91:68–73.

Hess B, Kafert S, Heinisch U, Wenger DA, Zlotogora J, Gieselmann V.1996. Characterization of two arylsulfatase A missense mutationsAsp335>Val and Thr274>Met causing late infantile metachromaticleukodystrophy. Hum Mutat 7:311–317.

Kafert S,HeinischU, Zlotogora J,GieselmannV. 1995.AmissensemutationP136L in the aylsulfatase A gene causes instability and loss of activity ofthe mutant enzyme. Hum Genet 95:201–204.

Kappler J, Sommerlade H, von Figura K, Gieselmann V. 1994. Complexarylsulfatase A alleles causing metachromatic leukodystrophy. HumMutat 4:119–127.

Kreysing J, von Figura K, Gieselmann V. 1990. Structure of thearylsulfatase A gene. Eur J Biochem 191:627–631.

Kreysing J, Bohne W, Bosenberg C, Marchesini S, Turpin JC, Baumann N,von Figura K, Gieselmann V. 1993. High residual arylsulfatase A(ARSA) activity in a patient with late-infantile metachromatic leukody-strophy. Am J Hum Genet 53:339–346.

Leinekugel P, Michel S, Conzelmann E, Sandhoff K. 1992. Quantitativecorrelation between the residual activity of b-hexosaminidase A andarylsulfatase A and the severity of the resulting lysosomal storagediseases. Hum Genet 88:513–523.

Marcao A, Amaral O, Pinto E, Pinto R, Sa Miranda MC. 1999. Metachro-matic leucodystrophy in Portugal—finding of four new molecularlesions: C300F, P425T, g.1190-1191insC, and g.2408delC. Hum Mut13:337–338.

Polten A, Fluharty AL, Fluharty CB, Kappler J, von Figura K, GieselmannV. 1991. Molecular basis of different forms of metachromatic leukody-strophy. N Engl J Med 324:18–22.

Stein C, Gieselmann V, Kreysing J, Schmidt B, Pohlmann R, Waheed A,Meyer H, O’Brien J, Von Figura K. 1989. Cloning and expression ofhuman arylsulfatase A. J Biol Chem 264:1252–1259.

von Bulow R, Schmidt B, Dierks T, Schwabauer N, Schilling K, Weber E,Uson I, von Figura K. 2002. Defective oligomerization of arylsulfatase Aas a cause of its instability in lysosomes and metachromatic leukody-strophy. J Biol Chem 277:9455–9461.

Von FiguraK, Steckel F, Hasilik A. 1983. Juvenile and adultmetachromaticleukodystrophy: partial restoration of arylsulfatase A (cerebrosidesulfatase) activity by inhibitors of thiol proteinases. Proc Natl Acad SciUSA 80:6066–6070.

Von Figura K, Gieselmann V, Jaeken J. 2001. Metachromatic leukodystro-phy. In: Scriver CR, et al., editor. The metabolic and molecular basisof inherited disease. 8th edition. New York: McGraw-Hill. p 3695–3724.

242 Marcao et al.