Palmitoyl-protein thioesterase (PPT) deficiency was recently shown to be the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The available enzyme assay is complicated and impractical for diagnostic use and is, in practice, unavailable. We have developed a new fluorimetric assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone. This PPT assay is simple, sensitive, and robust and will facilitate the definition of the full clinical spectrum associated with a deficiency of PPT. PPT activity was readily detectable in fibroblasts, leucocytes, lymphoblasts, amniotic fluid cells, and chorionic villi, but was profoundly deficient in these tissues from INCL patients. Similarly, a deficiency of PPT was shown in patients with the variant juvenile NCL with GROD. These results show that rapid pre- and postnatal diagnosis can be performed with this new enzyme assay for PPT.   Keywords: infantile neuronal ceroid lipofuscinosis; CLN1; palmitoyl-protein thioesterase; enzyme analysi

Catsman-Berrevoet..., C

Keulemans, J

Kleijer, W

Mancini, G

van Diggelen, O P

Voznyi, Y

Winchester, B

Young, E

PubMed

Voznyi, YV

Keulemans, JLM

Verheijen - Mancini, Grazia

Catsman - Berrevoets, Coriene

Diggelen, Otto

EUR Research Repository

 1999;36;471-474 J. Med. Genet.  Winchester, W J Kleijer and O P van Diggelen Y V Voznyi, J L M Keulemans, G M S Mancini, C E Catsman-Berrevoets, E Young, B   (INCL) and its variantsdiagnosis of infantile neuronal ceroid lipofuscinosis A new simple enzyme assay for pre- and postnatal http://jmg.bmj.com/cgi/content/full/36/6/471Updated information and services can be found at:  These include: References http://jmg.bmj.com/cgi/content/full/36/6/471#otherarticles2 online articles that cite this article can be accessed at:    http://jmg.bmj.com/cgi/content/full/36/6/471#BIBLThis article cites 11 articles, 5 of which can be accessed free at: Rapid responses http://jmg.bmj.com/cgi/eletter-submit/36/6/471You can respond to this article at:  serviceEmail alertingtop right corner of the article Receive free email alerts when new articles cite this article - sign up in the box at theTopic collections (3948 articles) Genetics   Articles on similar topics can be found in the following collections  Notes    http://www.bmjjournals.com/cgi/reprintformTo order reprints of this article go to:  http://www.bmjjournals.com/subscriptions/ go to: Journal of Medical GeneticsTo subscribe to  on 29 November 2006 jmg.bmj.comDownloaded from Short reportsA new simple enzyme assay for pre- and postnataldiagnosis of infantile neuronal ceroid lipofuscinosis(INCL) and its variantsY V Voznyi, J L M Keulemans, G M S Mancini, C E Catsman-Berrevoets, E Young,B Winchester, W J Kleijer, O P van DiggelenAbstractPalmitoyl-protein thioesterase (PPT) de-ficiency was recently shown to be the pri-mary defect in infantile neuronal ceroidlipofuscinosis (INCL). The available en-zyme assay is complicated and impracti-cal for diagnostic use and is, in practice,unavailable. We have developed a newfluorimetric assay for PPT based on thesensitive fluorochrome 4-methylumbelli-ferone. This PPT assay is simple, sensi-tive, and robust and will facilitate thedefinition of the full clinical spectrumassociated with a deficiency of PPT. PPTactivity was readily detectable in fibro-blasts, leucocytes, lymphoblasts, amnioticfluid cells, and chorionic villi, but wasprofoundly deficient in these tissues fromINCL patients. Similarly, a deficiency ofPPT was shown in patients with thevariant juvenile NCL with GROD. Theseresults show that rapid pre- and postnataldiagnosis can be performed with this newenzyme assay for PPT.(J Med Genet 1999;36:471–474)Keywords: infantile neuronal ceroid lipofuscinosis;CLN1; palmitoyl-protein thioesterase; enzyme analysisThe neuronal ceroid lipofuscinoses (NCL) area group of inherited, progressive encephalopa-thies characterised by lipofuscin-like inclusionsin various tissues. At least four distinct clinicalforms have been described. The infantile formof neuronal ceroid lipofuscinosis (INCL,Santavuori-Haltia disease, MIM 256730) is anearly onset, neurodegenerative disease charac-terised by psychomotor regression, seizures,and progressive macular degeneration leadingto blindness after the first year. Severe brainatrophy is observed by neuroimaging. Theinheritance of INCL is autosomal recessive andthe classical form is frequent in Finland (1 in13 000 births) and rare in the rest of the world.Electron microscopical investigations of skin orrectal biopsies show the characteristic granularosmiophilic deposits (GROD).Until recently, the confirmation and classifi-cation of the subtypes was based entirely on theappearance of the pathological inclusion bod-ies, being GROD, fingerprint-, or curvilinearstructures.1 During the last few years rapidadvances have been made in discovering theprimary defects in various forms of NCL. In1995 the gene involved in juvenile NCL(CLN3) was identified by the Batten diseaseconsortium.2 The corresponding protein is anintegral lysosomal membrane protein,3 but itsbiological role is still unknown. In 1997, Sleatet al4 showed that patients with the classicalform of late infantile NCL had mutations in agene (CLN2) which had homology to bacterialpepstatin insensitive carboxypeptidases. A cor-responding carboxypeptidase was found to bedeficient in brain extracts from late infantileNCL patients.In 1995, Vesa et al5 reported a deficiency ofpalmitoyl-protein thioesterase (PPT) in pa-tients with the infantile form of NCL andfound mutations in the corresponding CLN1gene. PPT is a typical soluble lysosomal hydro-lase which is routed to lysosomes through themannose-6-phosphate signal.6 This classifiesINCL as a genuine lysosomal storage disorderand has ended the debate about whether infan-tile NCL belongs to the group of lysosomalstorage disorders.The published enzyme assay for PPT7 uses a[3H]-S-palmitoyl-H-Ras protein as substrate.The substrate has been made in a baculovirusexpression system labelled in situ with [3H]-palmitate followed by elaborate purification.PPT hydrolyses the thioester linkage betweenthe palmitoyl group and the sulphur atom ofparticular cysteine residues from the H-Rasprotein. Unfortunately, this sophisticated assayis not practical for the diagnostic enzyme labo-ratory. Recently, a modification of this assayusing a [3H]-S-palmitoyl-peptide has beenpublished.8 We present here a novel and simpleassay for PPT based on the widely used fluoro-chrome 4-methylumbelliferone, show its appli-cation in rapid postnatal enzyme analysis, andreport the first, retrospective, prenatal enzymeanalyses of cases aVected with INCL.Patients and methodsFibroblasts and leucocytes were obtained frompatients with the typical clinical signs andJ Med Genet 1999;36:471–474 471Institute of OrganicChemistry, Moscow,RussiaY V VoznyiDepartment of ClinicalGenetics, UniversityHospital, ErasmusUniversity, PO Box1738, 3000 DRRotterdam,The NetherlandsJ L M KeulemansG M S ManciniW J KleijerO P van DiggelenDepartment of ChildNeurology, UniversityHospital, ErasmusUniversity, Rotterdam,The NetherlandsC E Catsman-BerrevoetsInstitute of ChildHealth, London, UKE YoungB WinchesterCorrespondence to:Dr van Diggelen.Received 13 August 1998Revised version accepted forpublication 15 December1998 on 29 November 2006 jmg.bmj.comDownloaded from symptoms of INCL. The diagnosis was con-firmed by the finding of GROD in appropriatetissues.The synthetic substrate for palmitoyl-proteinthioesterase (PPT) was prepared by linking4-methylumbelliferyl-6-thio-â-D-galactopyrano-side (MU-6-thio-â-galactoside) to palmitatethrough a thioester bond (MU-6S-Palm-âGal;available from Moscerdam Substrates, for in-quiries O P van Diggelen). Total leucocytes wereisolated from heparinised blood as describedpreviously9 and frozen until use. Skin fibroblastswere cultured according to routine proceduresin Ham’s F10 medium supplemented with 10%fetal bovine serum and antibiotics. The cellswere harvested with trypsin seven days after thelast subculture and stored at −70°C until use.Lymphoblasts were prepared by transformingperipheral blood lymphocytes with Epstein Barrvirus, according to standard procedures. Ho-mogenates were prepared by sonication of cellmaterial in water. For the standard one step PPTassay, reaction mixtures consisted of 10 µlhomogenate (2 µg protein for fibroblasts,lymphoblasts, and amniocytes; 5 µg for chori-onic villi and leucocytes) and 20 µl substratesolution containing 0.64 mmol/l MU-6S-Palm-âGal, 15 mmol/l dithiothreitol (DTT), 0.375 %(w/v) Triton X-100, and 0.1 U â-galactosidasefrom Aspergillus oryzae (Sigma) in McIlvain’sphosphate/citrate buVer, pH 4.0. The reactionmixtures were incubated for 17 hours at 37°C.The exogenous â-galactosidase from Aspergilluswas necessary since the endogenousâ-galactosidase activity was insuYcient to con-vert the reaction intermediate MU-6-thio-â-galactoside quantitatively. PPT activities weresimilar in acetate buVer and phosphate/citratebuVer. Mg2+ and EDTA (7 mmol/l) had no effecton the PPT activity.In the two step assay, the first incubation wasidentical to the standard assay, except thatAspergillus â-galactosidase was not added. Afterthe first incubation of 17 hours at 37°C, thesamples were boiled for two minutes to stop thePPT reaction. After cooling, 0.1 U of Aspergil-lus â-galactosidase was added and the secondstep was incubated for six hours at 37°C tohydrolyse the reaction intermediate MU-6-thio-â-galactoside. In the experiment to deter-mine the optimum pH, buVer was added toadjust the pH to 4.5. Finally, all enzymaticreactions were terminated by the addition of200 µl of 0.5 mol/l Na2CO3/NaHCO3, pH 10.7and the fluorescence of 4-methylumbelliferone(MU) was measured in a FluoroCount (Pack-ard) fluorimeter. Protein was determined asdescribed previously.9ResultsThe potential of 4-methylumbelliferyl-6-thiopalmitoyl-â-D-galactopyranoside (MU-6S-Palm-âGal) as an artificial substrate for thepalmitoyl-protein thioesterase (PPT) was in-vestigated. The liberation of MU from MU-6S-Palm-âGal cannot be accomplished by the soleaction of PPT, since this enzyme only hydro-lyses the palmitoyl thioester linkage, yieldingthe non-fluorescent reaction intermediateMU-6-thio-â-galactoside. The endogenousâ-galactosidase, present in cell homogenates,was not able to hydrolyse the reaction interme-diate quantitatively, but exogenousâ-galactosidase from Aspergillus (0.1 U) couldhydrolyse MU-6-thio-â-galactoside completelywithin six hours, albeit at a low rate (data notshown). The release of MU required the pres-ence of a reducing agent (DTT) to prevent for-mation of disulphides between MU-6-thio-â-galactosides. Using the two step assay (seePatients and methods section), the optimumpH of PPT for leucocytes and fibroblasts wasshown to be 4 (fig 1A). Activities at pH >6could not be determined owing to spontaneoushydrolysis of the palmitoyl thioester bond ofMU-6S-Palm-â-Gal. The pH optima for am-niocytes and chorionic villi were similar (notillustrated). In fibroblasts and leucocytes fromINCL patients, the thioesterase activity at pH3.5 was almost absent but gradually increasedat higher pH values, reaching control levels atpH 6. This indicated that thioesterases otherthan PPT can also hydrolyse the substrate athigher pHs. All further experiments were doneat pH 4. The PPT assay could also be carriedout in one step by adding exogenousâ-galactosidase together with the substrate. Infig 1B, PPT activities in fibroblasts and leuco-cytes, measured in one and two steps, are com-pared. In the two step assay, diVerent incuba-tion times for the first (PPT) step werefollowed by an incubation period of six hourswith exogenous â-galactosidase. In the one stepassay, activities are underestimated if incuba-Figure 1 (A) pH dependence of palmitoyl-protein thioesterase activity. Fibroblasts from a control (open triangles) and an INCL patient (filled triangles);leucocytes from a control (open squares) and an INCL patient (filled squares). (B) Time course of palmitoyl-protein thioesterase activity in the one stepassay (closed symbols) and two step assay (open symbols). Control fibroblasts (triangles) and control leucocytes (squares). (C) Protein dependence ofpalmitoyl-protein thioesterase activity. Control fibroblasts (triangles), control leucocytes (squares), control amniocytes (diamonds), control chorionic villi(circles). Reaction conditions as described in the Patients and methods section, except for the parameter which is varied.2000400pHAnmol/17 h/mg3.56004.0 4.5 5.0 5.5 6.0 2500500Incubation time (h)BActivity nmol/mg075001252504 8 12 16203Protein (µg)CActivity nmol/mgActivity (nmol/17 h)0144 6 82472 Voznyi, Keulemans, Mancini, et al on 29 November 2006 jmg.bmj.comDownloaded from tion times are less than four hours owing to theslow hydrolysis of the reaction intermediateMU-6-thio-â-galactoside by â-galactosidase.However, after an overnight incubation, theone step assay approached the “true” PPTactivity as measured in the two step assay.Therefore, a one step assay with a 17 hourincubation was used in the experimentsdescribed below. Amniocytes and chorionicvilli gave similar results (not shown). The PPTactivity increased in a non-linear manner withthe amount of protein up to 8 µg (fig 1C). As acompromise between linearity and sensitivityof the assay, we chose the fixed proteinamounts as mentioned in the Patients andmethods section. The apparent Km wasestimated to be 0.2 mmol/l for fibroblasts andleucocytes (data not shown).Under standard conditions PPT activity wasreadily detectable in control leucocytes, lym-phoblasts, fibroblasts, amniocytes, and chori-onic villi (table 1), whereas the activity wasreduced in leucocytes and fibroblasts fromobligate heterozygotes for INCL. PPT activitywas profoundly deficient in fibroblasts, totalleucocytes, and lymphoblasts from patientswith INCL (table 1). PPT activity was alsodeficient in leucocytes from the variant juvenileNCL with GROD. In fibroblasts from patientswith I cell disease, a multiple lysosomal enzymedeficiency, PPT was considerably reduced.Retrospectively, a clear deficiency of PPTwas shown in amniotic fluid cells (amniocytes)and chorionic villi from fetuses aVected withINCL (table 1). This shows the feasibility ofprenatal enzyme analysis for INCL.DiscussionWe have synthesised an artificial substrate forpalmitoyl-protein thioesterase with a thioesterbond between palmitate and 6-thiogalactoselinked to the fluorochrome 4-methylumbelli-ferone (MU-6S-Palm-âGal). The specificity ofthis substrate was shown by the deficiency ofPPT in various cell types from patients with theinfantile form of neuronal ceroid lipofuscinosisand its variants. To our knowledge, thioestersbetween fatty acids and thio-sugars do not existin nature and our results show that PPT doesnot discriminate between the compounds towhich the fatty acid thioester is linked. This isconsistent with the observation that PPT alsohydrolyses palmitoyl-CoA.7 Thus, PPT sharesa property typical of most lysosomal enzymes,viz that it is the terminal group of the linkagehydrolysed by the enzyme that determines thespecificity of the enzyme. This property hasbeen exploited for diagnostic purposes andnearly all the lysosomal storage disorders arenow diagnosed by enzyme assays using artifi-cial substrates based on the sensitive fluoro-chrome 4-methylumbelliferone.Since the primary defect in infantile NCLwas reported,5 it has become clear that classifi-cation based on age of onset and electronmicroscopical histopathology is no longercompletely reliable and that a heterogeneousclinical phenotype of a deficiency of PPT isemerging. Das et al10 showed that only half ofthe patients with PPT deficiency in the USAand Canada have the severe phenotype charac-teristic of the Finnish INCL. The availability ofa simple enzyme assay will greatly facilitate thedefinition of the full clinical spectrum of a defi-ciency of PPT and it is an intriguing thoughtthat late onset patients with PPT deficiency,with or without GROD, might be discovered byenzyme analysis. The correlation between thepresence of GROD and a deficiency of PPT isgood but not absolute. A patient with GRODin ganglion cells, but homozygous for amutation in the gene for classical juvenile NCL(CLN3) has been reported.11In the Finnish population, screening for justa single mutation has been an eYcient meansof confirming the diagnosis in 95% of theINCL patients,5 albeit that the sensitivity is not100%. In non-Finnish patients with a PPTdeficiency, a large number of diVerent muta-tions has been found,10 indicating that screen-ing for known mutations is not eYcient inidentifying the NCL subtype. In contrast, thepresent assay of functional PPT screens for allknown and unknown mutations leading to aPPT deficiency. Therefore, enzyme analysisshould always precede mutation analysis in theprocess of establishing the diagnosis in newpatients. In fact, enzymatic confirmation ofPPT deficiency in Dutch patients suspected ofhaving INCL has initiated a screening pro-gramme for mutations in the CLN1 gene inThe Netherlands.Our fluorogenic PPT assay is much moreconvenient than the assays using purified[3H]-palmitoylated-H-Ras protein7 or a [3H]-palmitoylated peptide.8 We have shown that thepresent PPT assay is a straightforward way toconfirm the diagnosis of patients suspected ofhaving INCL. We have also shown that it ispossible to see a deficiency of PPT in fetalsamples.We thank Professor Hans Galjaard for his continuing supportand Tom de Vries Lentsch for photography. This work was sup-ported by INTAS (International Association for the Promotionof Co-operation with Scientists from the former Soviet Union,project 94-3625). We thank Professor Pirkko Santavuori formaking available blood and fibroblasts from Finnish INCLpatients and Dr Irene Hofman for providing blood from patientswith vJNCL with GROD. We also thank Dr D J van Rhenen andthe staV of the “Bloedbank, Rotterdam” for providing indispen-sable blood samples from controls.1 Goebel HH. The neuronal ceroid-lipofuscinoses. J ChildNeurol 1995;10:424-37.2 The International Batten’s Disease Consortium. Isolation ofa novel gene underlying Batten disease CLN3. Cell1995;82:949-57.Table 1 PPT deficiency in infantile and variant juvenile ceroid lipofuscinosisPalmitoyl-protein thioesterase activity (nmol/17h/mg)Fibroblasts Leucocytes Lymphoblasts AmniocytesChorionicvilliINCL Finnish 7–21 2; 10(n=5) (n=2)INCL non-Finnish 5–22 4–13 6 20 18(n=12) (n=4) (n=1) (n=1) (n=1)vJNCL+GROD 7; 9(n=2)INCL heterozygotes 399–435; 640 99–128(n=5) (n=5)I cell disease 45(n=1)Controls 380–850 90–280 260–450 410–910 125–520(n=32) (n=48) (n=5) (n=12) (n=15)Pre- and postnatal enzyme analysis for INCL 473 on 29 November 2006 jmg.bmj.comDownloaded from 3 Järvelä I, Sainio M, Rantamaki T, et al. Biosynthesis andintracellular targeting of the CLN3 protein defective inBatten disease. Hum Mol Genet 1998;7:85-90.4 Sleat DE, Donnelly RJ, Lackland H, et al. Association ofmutations in a lysosomal protein with late-infantile neuro-nal ceroid lipofuscinosis. Science 1997;277:1802-5.5 Vesa J, Hellsten E, Verkruyse LA, et al. Mutations in thepalmitoyl protein thioesterase gene causing INCL. Nature1995;376:584-7.6 Verkruyse LA, Hofmann SA. Lysosomal targeting ofpalmitoyl-protein thioesterase. J Biol Chem 1996;271:15831-6.7 Camp LA, Hofman SA. Purification and properties of apalmitoyl-protein thioesterase that cleaves palmitate fromH-Ras. J Biol Chem 1994;268:22566-74.8 Cho S, Dawson G. Enzymatic and molecular biologicalanalysis of palmitoyl-protein thioesterase deficiency ininfantile neuronal ceroid lipofuscinosis. J Neurochem 1998;71:323-99 van Diggelen OP, Zhao H, Kleijer WJ, et al. A fluorometricenzyme assay for the diagnosis of Morquio disease type A(MPS IV A). Clin Chim Acta 1990;187:131-40.10 Das KD, Becerra CHR, Yi W, et al. Molecular genetics ofpalmitoyl-protein thioesterase deficiency in the U.S. J ClinInvest 1998;102:361-70.11 Åberg L, Järvelä I, Rapola J, et al. Atypical juvenile neuronalceroid lipofuscinosis with granular osmiophilic deposit-likeinclusions in the autonomic nerve cells of the gut wall. ActaNeuropathol 1998;95:306-12.474 Voznyi, Keulemans, Mancini, et al on 29 November 2006 jmg.bmj.comDownloaded from 

English

A new simple enzyme assay for pre- and postnatal diagnosis of infantile neuronal ceroid lipofuscinosis (INCL) and its variants

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Palmitoyl-protein thioesterase (PPT) deficiency was recently shown to be
          the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The
          available enzyme assay is complicated and impractical for diagnostic use
          and is, in practice, unavailable. We have developed a new fluorimetric
          assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone.
          This PPT assay is simple, sensitive, and robust and will facilitate the
          definition of the full clinical spectrum associated with a deficiency of
          PPT. PPT activity was readily detectable in fibroblasts, leucocytes,
          lymphoblasts, amniotic fluid cells, and chorionic villi, but was
          profoundly deficient in these tissues from INCL patients. Similarly, a
          deficiency of PPT was shown in patients with the variant juvenile NCL with
          GROD. These results show that rapid pre- and postnatal diagnosis can be
          performed with this new enzyme assay for PPT

A new simple enzyme assay for pre- and postnatal diagnosis of infantile neuronal ceroid lipofuscinosis (INCL) and its variants

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