P-Galactosidase in Tissue Culture Derived from Human Skin and Bone Marrow: Enzyme Defect in Gmlgangliosidosis

Pediat. Res. 3: 532-537 (1969) Fibroblasts GM1gangliosidosis bgalactosidase gangliosides

P-Galactosidase in Tissue Culture Derived from Human Skin and Bone Marrow: Enzyme Defect in GMlGangliosidosis

HOWARDR. SLOAN[~", B. WILLIAMUHLENDORF, CECIL B. JACOBSON and DONALDS. FREDRICKSON

Molecular Disease Branch, National Heart Institute, and Laboratory of Viral Immunology, Division of Biologics Standards, Bethesda, Maryland, and Department of Obstetrics and Gynecology, George Washington University School of Medicine, Washington, D.C.

Extract

B-Galactosidase activities were determined in fibroblast preparations derived from the bone marrow and skin of a patient with GM~gangliosidosis and from 11 control subjects. Two B-galactosidase substrates were employed in these studies: ganglioside GMI,labeled with tritium in the terminal galactose by a new procedure, and o-nitrophenyl-P-galactopyranoside. The GMI-B-galactosidase activity in the cells of the patient was reduced 17- to 30-fold compared with the activity in the cells of the control subjects; also, the cells of the patient exhibited an 11- to 30-fold depression in o-nitro- phenyl-B-galactosidase activity (ONP-B-galactosidase). These results demonstrated that fibroblasts cultured from an affected subject might be employed for diagnostic purposes. The GMI-and o-nitro- phenyl-B-galactosidase activities of fibroblasts cultured from normal amniotic fluid have also been determined; o-nitrophenyl-B-galactosidase activities ranged from 61 to 153 units/106 cells and the GMI-8-galactosidase activities varied between 45 and 95 units/ 1O8 cells.

Speculation

Determination of GMI-B-galactosidase activity in fibroblasts cultured from skin may permit the de- tection of individuals heterozygous for GMIgangliosidosis. The presence of a p-galactosidase capable of releasing the terminal galactose from ganglioside GMIin fibroblasts derived from amniotic fluid offers a basis for possible intrauterine diagnosis of GM~gangliosidosis.

Introduction [(2 + 3)-N-acetylneuraminyll-galactosyl-(1 + 4)- glucosyl-(1 + 1)-[2-N-acyll-sphingosine,in the brain Generalized gangliosidosis (GMl gangliosidosis) is a and visceral organs [9, 11,221. Clinically, the disorder rare inherited metabolic disorder characterized by the is manifested by progressive neurological deterioration, accumulation of large quantities of GMl ganglioside, accumulation of glycolipid in neurons and reticulo- galactosyl-(1 + 3)-N-acetyl-galactosaminyl-(I+ 4)- endothelial cells, skeletal and facial features similar to ~RSONand FREDRICKSON 533 those seen in Hurler's syndrome, and death by 2 years of' harvesting the cells were rinsed with Ca2+, Mg2+- of age. DERRYet al. [3] have proposed that there is a free Dulbecco's saline solution [4] and removed from second form of GMl gangliosidosis in which ganglio- the glass by treatment with this solution containing: side GM1(GMl) accumulates only in the brain without (in percent) trypsin, 0.05; ethylenediaminetetraacetate osseous defects or visceromegaly. (EDTA), 0.005; and methylcellulose, 0.2. Cells, 30-60 A deficiency of o- and p-nitrophenyl-j3-galactosidase x 106, were then suspended in 40 ml of Dulbecco's activity has recently been demonstrated in the liver, saline containing 0.1 % methylcellulose, and a 0.5-ml spleen, kidney, and brain of four patients with GMl aliquot was removed for counting [28]. The cell sus- gangliosidosis [2, 12, 151. In addition, OKADAand pension was then centrifuged at 600 x g; the pellet was O'BRIEN[I21 demonstrated depressed levels of GM~- resuspended in 10 ml of cold methylcellulose-saline j3-galactosidase activity in tissues from two of these and centrifuged at 600 xg. The cell pellet was stored patients. Deficiency in the activity of lysosomal 8- at -80' until processed further. galactosidase, which normally cleaves the terminal Other cells were suspended in 1 ml of 0.25 M sucrose galactose from the oligosaccharide moiety of GM1, containing 2.5% Cutscum (isooctylphenoxypolyoxy- probably accounts for the accumulation of GMl in ethanol) [29] and disrupted by the microtip of a soni- generalized gangliosidosis. fier [30]. The output control selector was set at no.2 We have determined the j3-galactosidase activity and the output of the microtip was 1.2 amp. The cells with o-nitrophenyl-j3-D-galactopyranoside (ONPG) as were sonified at 0' for two 30-sec periods, interrupted substrate in fibroblast cultures derived from the skin by a 1-min pause. Aliquots of the resulting preparation and from bone marrow of a patient with GMlganglio- were assayed immediately or after storage at -70'. Re- sidosis and in cultures from 11 control subjects. An peated freezing and thawing did not affect the activity additional method using tritiated GMl (GM1m3H)was of the various enzymes assayed. also developed to assay the enzyme. The incubation mixture for the assay of ONP-j3- galactosidase activity contained an aliquot of the fibro- blast preparation representing approximately lo5 Subjects and Methods cells; 25 pmoles of sodium acetate buffer, pH 5.1; 1.33 pmoles of ONPG; 200 pg of Cutscum; and water Patient MB (NIH 07-22-86) was 14 months old at the in a final volume of 0.2 ml. After incubation for 90 min time the biopsies for initiation of cell cultures were at 37O, the reaction was terminated by the addition of obtained; he died at the age of 2 years. The diagnosis 0.1 ml of human serum albumin (100 mg/ml) and 0.5 of GMlgangliosidosis was established by isolating and ml of 4% trichloroacetic acid. The clear supernatant quantifying the gangliosides from portions of brain solution obtained by low speed centrifugation (500 x g) and liver 110, 221. These tissues contained 1,900 and was pipetted into 0.4 ml of 1 M sodium carbonate. 108 pg of GM~sialic acid/g wet weight, respectively, The o-nitrophenol concentration was determined by an increase of 20-50 times the normal GM1content measuring the optical density at 415 mp. One unit of [3, 19, 201. ~~~-/?-~alactosidaseactivity was defined as the Control cells were derived from one normal subject amount of enzyme required to catalyze the hydrolysis and from patients with diseases other than GM1gang- of 1 nmole (nanomole) of ONPG/h under the standard liosidosis. The control subjects ranged in age from conditions. 6 months to 26 years of age. Cultures were initiated GMI, isolated from the brain of patient MB, was from bone marrow aspirates and from 3-mm punch tritiated by oxidation with galactose oxidase followed skin biopsies [23]. The growth medium used was by reduction with sodium borohydride-3H (17). The Eagle's 'minimum essential medium' containing the radioactive GMl was purified by column chromato- nonessential amino acids [5], 10% fetal bovine serum graphy on Anasil S [I 31. The purified GMI-~Hmigrat- and neomycin (50 pg/ml). Usually in 3 weeks sufficient ed as a single band on thin-layer plates of silica gel G outgrowth of fibroblasts from primary explants oc- that were developed in either chloroform-methanol- curred to permit subcultivation of the cells at 3- to 2.5 N ammonium hydroxide (60 : 35 :8) or n-propanol- 6-day intervals. The cells used for subcultivation were water (7:3) [27]. The labeled ganglioside had the removed from the glass surface and dispersed with same RF as authentic GMl. Incubation of the GM1-3H 0.25 % trypsin. As viewed under the light microscope, with rat brain j3-galactosidase [7] demonstrated that the cell morphology and growth pattern of the cells more than 90% of the tritium was in the terminal from the patient with GM~and those from the control galactose moiety. subjects were the same. GMvrl-"was used as a specific substrate for the deter- Cells used for enzyme assays were grown as mono- mination of GM1-8-galactosidase activity. The incu- layer cultures in 32-02 prescription bottles. At the time bation mixture contained an aliquot of the fibroblast 534 SLOAN,UHLENDORF, JACOBSON and FREDRICKSON

Table I. j3-Galactosidase activities in fibroblasts cultured from bone marrow and skin biopsies of control subjects ' and of a patient with GM1gangliosidosis

Patients Age Type of Diagnosis j3-Galactosidase Activity of other biopsy activity with lysosomal substrate enzymes ONPG1 G- Acid j3-N- 3H phos- acetyl- phatase3 galactos- amini- dase4 Control cultures 8 years Marrow Inclusion body encephalitis 4 years Marrow Niemann-Pick disease 2 years Marrow Niemann-Pick disease 18 months Marrow Undifferentiated lipidosis 15 months Marrow Mental retardation 6 months Marrow Niemann-Pick disease 59 years Skin Rheumatic heart disease 22 years Skin Niemann-Pick disease 21 years Skin Normal volunteer 5 years Skin Vogt-Spielmeyer disease 18 months Skin Niemann-Pick disease 15 months Skin Mental retardation Patient's cultures MB 18 months Marrow GMlgangliosidosis 6 < 3 464 43 MB 18 months Skin GMl gangliosidosis 3 < 3 398 39

Activity expressed as nmoles o-nitrophenyl-b-D-galactopyranosidehydrolyzed/106 cells/h. Activity expressed as nmoles galactose-3H released/108 cells/h. Activity expressed as nmoles p-nitrophenylphosphate hydrolyzed/106 cells/h. 4 Activity expressed as nmoles p-nitrophenyl-b-N-acetyl-galactosaminidehydrolyzed/lOO cellslh.

preparation representing approximately lo5 cells; 25 galactosidase activity as measured with ONPG or pmoles of sodium acetate buffer, pH 5.1; 2.5 nmoles GMVr1-3Hwas linear with respect to time and enzyme of GMl-3H(17 mCi/mmole) ; 1 pmole sodium deoxy- concentration over the range examined. taurocholate [29]; 200 pg of Cutscum; and water in Acid phosphatase and B-N-acetyl-galactosaminidase a final volume of 0.2 ml. activities of the tissue culture preparations were also After incubation for 90 min at 37O, the reaction was determined [8, 181. The substrates, p-nitrophenyl- terminated by heating for 3 min at 100'. The galactose phosphate and p-nitrophenyl-j3-N-acetylgalactosami- liberated fromG~1was separated from excess GMland nide, were obtained commercially [32]. the other reaction product, ganglioside GMl [31], by thin-layer chromatography. Chromatography was performed on thin-layer plates of silica gel G with Results and Discussion chloroform-methanol-2.5 N ammonium hydroxide (60: 35 :8) as solvent. The area corresponding to galac- A deficiency of both ONP-j3-galactosidase and ganglio- tose was scraped and counted in a liquid scintillation side GM1-/3-galactosidaseactivities was found in fibro- counter with a Triton-toluene counting fluid [I]. One blast cultures derived from skin and from bone marrow unit of GM1-p-galactosidase activity was defined as biopsies of patient MB (table I). The ONP-Pgalacto- the amount required to catalyze the release of 1 nmole sidase activity in his cultures was 2.5-10% of the activ- galactose-3H/h under the standard conditions. The ity in the cultures from control subjects. With GMl-3H /3-Galactosidase in tissue cultures from human skin and bone marrow 535 as substrate, cell cultures of patient MB demonstrated dilution of the labeled substrate by endogenous GM~ no activity by an assay capable of detecting 3 units of and not from the presence of an inhibitor in the pa- activity per lo8cells; GM1-B-galactosidaseactivity was tient's cells. less than 6% of that in the least active control culture. The marked difference between the ONP- and The deficiency of activity against either galactoside GMl-8-galactosidase activities in the cells of the control was specific for GM1gangliosidosis (table I). subjects was probably the result of the different solubil- The activities of two other lysosomal hydrolases, ity characteristics of the two substiates. ONPG is water acid phosphatase and j3-N-acetylgalactosaminidase, soluble and, therefore, probably more available to were determined to demonstrate the metabolic com- the enzyme than GM1,which presumably was present petency of the cells from patient MB. The activities of in micellar form. Further metabolism of the galactose these enzymes were not depressed (table I). Aliquots liberated from GM1would also result in a lower appar- of a single sonically disrupted cell preparation were ent GMl-B-galactosidaseactivity. The latter possibility used for multiple /3-galactosidase and 'control' enzyme is probably of minor importance, however, since more assays. In most cases the values in table I are the means than 90% of the GM1could be recovered following of values from two or three assays. In three subcultures incubation. of skin and two of the bone marrow, cells of patient MB We have also cultured a number of cell lines from exhibited low levels of ONP- and GM1-8-galactosidase the fetal cells present in amniotic fluids, obtained by activity. transabdominal amniocentesis or at therapeutic abor- The results of assays for ONP- and GMl-/3-galact0si- tion after 12-20 weeks of gestation. ONP-j3-galactosi- dase activities in control cells with and without ad- dase and GMl-/I-galactosidase activities were assayed mixture of cells from the patient are shown in table 11. in four of these lines. The ONP-/3-galactosidase activi- Cells from the patient did not inhibit ONP-j3-galacto- ties ranged from 61 to 153 units/106 cells; the G~i-j3- sidase activity; however, there appeared to be a de- galactosidase activities varied between 45 and 95 crease in the GM1-j3-galactosidaseactivity of the con- units/108 cells [24]. Thus, cultures derived from am- trol cells in the presence of cells from patient MB. This niotic fluid may make possible the detection of GM~ depressed activity was eliminated by tripling the con- gangliosidosis in the developing fetus as early as week centration of GMl-3H in the incubation mixtures. 22 of gestation. These experiments indicate that the lower j3-galacto- Sodium borohydride-3H, with a specific radioactivity sidase activities in the mixtures probably resulted from of 400 mCi/mmole, was used in the preparation of the GMl-3H,and yielded a labeled substrate that permitted the determination of the GMl-j3-galactosidaseactivity in lo5 fibroblasts. Sodium borohydride-3H is now Table II. /3-Galactosidase activity in mixtures of cells available with a specific radioactivity of 6 Ci/mmole. from control subjects and patient MB If this reagent was employed, a 15-fold increase in the specific radioactivity of GMl-3H could be obtained. Control Type of Enzyme activity in cells from Use of this substrate and prolonged incubation of the subjects cell Control alone Control+ MB1 assay might permit the determination of the GM1-b- galactosidase activity in cultured cells as early as 2 ONP-B-galactosidase weeks after the amniotic fluid was obtained. PO Skin 63 59 After this work was completed, PINSKYet al. [14] PU Skin 81 78 reported depressed levels of ONP-/?-galactosidase AS Bone marrow 80 83 activity in cultured fibroblasts of a patient in which GM1accumulated only in the brain. GMl-/3-galactosidase3 'Generalized' and 'cerebral' GMlgangliosidosis may AS Bone marrow 80 71 represent two manifestations of homozygosity for the VW Bone marrow 95 78 same defective gene. The amounts of visceral muco- VW Skin 57 46 polysaccharides and visceral glycolipids are two differ- -- ences in the chemical pathology of these two forms of 1 For each incubation 2-4 x lo5control cells were used; the disorder [2 11. It seems possible that these two forms in the mixtures 5 x 105 cells from patient MB were of GMl gangliosidosis may actually result from two added. different mutations at the p-galactosidase locus. Activities expressed as nmoles ONPG hydrolyzed/106 Compared with the normal enzyme, the mutant control cells/h. enzymes might possess different relative affinities for 3 Activities expressed as nmoles galactose-3H released/ GMl and mucopolysaccharides. Alternatively, one lo8 control cellslh. mutation might lead to a more severe deficiency of 536 SLOAN,UHLENDORF, JACOBSON and FREDRICKSON enzymatic activity and, therefore, to a more generaliz- 13. PENICK,R. J.; MEISLER,M.H. and MCCLUER, ed accumulation of GM1. The ready accessibility of R. H. : Thin-layer chromatographic studies of this enzymatic activity in cultured fibroblasts may human brain gangliosides. Biochim. biophys. Acta assist in the further assessment of these two possibilities. 116: 279 (1966). It is also entirely possible that enzymatic analysis of 14. PINSKY,L.; CALLAHAN,J.W. and WOLFE,L.S.: cultured fibroblasts may permit detection of the hetero- Fucosidosis? Lancet ii: 1080 (1968). zygous state in GM1gangliosidosis. 15. SACREZ,R.; JUIF,J. G.; GIGONNET,J. M. et GRU- NER, J.E.: La maladie de Landing ou idiotie amaurotique infantile prCcoce avec gangliosidose gCnCraliste de type GM1. Ptdiatrie 22: 143 References and JVO~~J (1967). 16. SELLINGER,O.Y.; BEAUFAY,H.; JACQUES, P.; 1. BENSON,R. H. : Limitations of tritium measure- DOYEN,A. and DEDUVE,C. : Tissue fractionation ments by liquid scintillation counting of emulsions. studies. 15. Intracellular distribution and pro- Analyt. Chem. 38: 1353 (1966). perties of p-N-acetylglucosaminidase and p-galac- 2. DACREMONT,G. and KINT,J.A.: Ghfl ganglioside tosidase in rat liver. Biochem. J. 74: 450 (1960). accumulation and ,!I-galactosidase deficiency in a 17. 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FROHWEIN,Y.Z. and GATT,S.: Isolation of @-N- (1968). acetylhexosaminidase, 8-N-acetylglucosaminidase, 20. SUZUKI,K.; SUZUKI,K. and CHEN,G. C. : Morpho- and ,!?-N-acetylgalactosaminidasefrom calf brain. logical, histochemical and biochemical studies on Biochemistry 6: 2775 (1967). a case of systemic late infantile lipidosis (general- 7. GATT,S.: Enzymatic hydrolysis of sphingolipids. ized gangliosidosis) . J.Neuropath. exp. Neurol. 27: V. Hydrolysis of monosialoganglioside and hexo- 15 (1968). sylceramides by rat brain /%galactosidase. Biochim. 2 1. SUZUKI,K. ;SUZUKI, K. and KAMOSHITA,S. : Chem- biophys. Acta 137: 192 (1967). ical pathology of GM1-gangliosidosis(generalized 8. GATT, S. and RAPPORT,M.M.: Isolation of ,!?- gangliosidosis). J.Neuropath. exp. Neurol. 28: 25 galactosidase and ,!?-glucosidase from brain. Bio- (1969). chim. biophys. Acta 113: 567 (1966). 22. SVENNERHOLM,L. : The gangliosides. J. Lipid Res. 9. GONATAS,N. K. and GONATAS,J. : Ultrastructural 5: 145 (1964). and biochemical observations on a case of systemic 23. UHLENDORF,B.W.; HOLTZ,A.I.; MOCK,M.B. late infantile lipidosis and its relationship to Tay- and FREDRICKSON,D. S. : Persistence of a metabolic Sachs disease and gargoylism. J. Neuropath. exp. defect in tissue cultures derived from patients with Neurol. 24: 318 (1965). Niemann-Pick disease; in: S. M. ARONSONand 10. KANFER,J.N. ;BLACKLOW, R. S. ;WARREN, L. and B.W.VOLK: Inborn disorders of sphingolipid me- BRADY,R. 0. : The enzymatic synthesis of ganglio- tabolism, p. 443 (Pergamon Press, New York 1966). sides. I. The incorporation of labeled N-acetyl- 24. UHLENDORF,B.W. ; SLOAN,H. R. ;JACOBSON,C. B. neuraminic acid into monosialoganglioside. Bio- and FREDRICKSON,D. S. (In preparation.) chem.Biophys. Res. Commun. 14: 287 (1964). 25. WATTIAUX,R.; WIBO,M. and BAUDHUIN,P.: In- 11. O'BRIEN, J. S.; STERN,M.B.; LANDING,B.H.; fluence of injection of triton WR-1339 on pro- O'BRIEN,J. 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27. WHERRETT,J. R. and CUMMINGS,J. N. : Detection minus the terminal galactose. and resolution of gangliosides in lipid extracts by 32. Mann Research Laboratories, 136 Liberty Street, thin-layer chromatography. Biochem. J. 86: 378 New York, N.Y. (1963). 33. We thank Dr. LYASINGER, Children's Hospital of 28. Coulter Electronics Inc., Hialeah, Fla. Washington, D.C., for providing the autopsy 29.,Cutscum was purchased from Fisher Scientific material on the patient. We thank Mrs. BARBARA Company, Fair Lawn, N. J., USA; sodium deoxy- DAVIS,Miss SENIYETEMEL, and Mrs. KRISTIN taurocholate was obtained from Maybridge Re- HINDSfor technical assistance. search Chemicals, Tintagel, North Cornwall, UK. 34. Requests for reprints should be addressed to: Ho- 30. Branson Instruments Inc., Stamford, Conn. WARD R.SLOAN,M.D., Ph.D., National Heart 3 1. The structure of GM~is identical to that of GMl Institute, Bethesda, Maryland 200 14 (USA).