Am J Hum Genet 30:644-652, 1978

The Nature of Mutation in

YOAV BEN-YOSEPH,1 MELINDA HUNGERFORD, AND HENRY L. NADLER

Krabbe disease (globoid cell ) is a disorder of galactolipid metabolism, inherited in an autosomal recessive manner [1]. Onset in early infancy is followed by rapidly progressive degeneration of the central nervous system, ending in death before the age of 2 years. The primary defect has been ascribed to a deficiency of /3-galactosidase activity [2]. In addition, a deficiency of /3- galactosidase activities towards psychosine (galactosylsphingosine) [3], monogalac- tosyl diglyceride [4], and lactosylceramide [5] have been reported. The inability to degrade these substrates, found in tissues of children who died of Krabbe disease, indicates that the same enzyme may be acting on the four galactolipids. These activities are, however, normal in patients with GM, gangliosidosis. Hydrolysis of lactosyl- under different assay conditions [6] and /3-galactosidase activities towards GM, , asialo-GMl ganglioside, and asialofetuin (ASF) are deficient in GM, gangliosidosis, but normal in Krabbe disease [7]. Thus, the mutation in Krabbe disease is not allelic with the GM, gangliosidosis mutation, and the observation that antibodies to GM, /3-galactosidase do not precipitate the activities of galactosylceramide 38- galactosidase [8, 9] supports the conclusion that these two /3-galactosidase enzymes are different proteins coded by separate loci. The present study defines the nature of the mutation in Krabbe disease. Using antibodies evoked against the normal galactosylceramide ,3-galactosidase enzyme purified from placenta, we found normal quantities of antigenically cross reacting material of the inactive mutant enzyme in brain, liver, and skin fibroblasts of patients with Krabbe disease. MATERIALS AND METHODS Galactose oxidase was obtained from Worthington Biochemical (Freehold, N.J.), Sepharose 4B from Pharmacia (Piscataway, N.J.), agarose from Aldrich Chemical (Milwaukee, Wis.), Coomassie Brilliant Blue G-250, acrylamide, N, N-methylene bis acrylamide, ammonium persulfate, TEMED and sodium dodecyl sulfate (SDS) from Eastman-Kodak (Rochester, N.Y.), DEAE-cellulose (Cellex-D), Bio beads SM-2 and Bio Gel A 1.5 m from Bio-Rad (Richmond, Calif.), and Eagles minimal essential medium (MEM) from Gibco (Grand Island, N.Y.).

Received January 27, 1978; revised May 22, 1978. This work was supported by grants from the National Foundation-March of Dimes and the Kroc Foundation. ' All authors: Department of Pediatrics, Northwestern University Medical School, Division of Genetics, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, Illinois 60614. © 1978 by the American Society of Human Genetics. All rights reserved. 644 NATURE OF MUTATION IN KRABBE DISEASE 645 Concanavalin A, dihydrosphingosine, N-stearoyl-dihydrogalactocerebroside, N-stearoyl- dihydrolactocerebroside, and goat antiserum to rabbit immunoglobulin G (IgG) were from Miles Laboratories (Elkhart, Ind.). 4-Methylumbelliferyl-,f-D-glucoside, 4-methylumbelliferyl-N- acetyl-,8-D-glucosaminide, and bovine serum albumin were from Sigma (St. Louis, Mo.), and naphthol AS-Bl-,8-D-galactoside was from Research Products International (Elk Grove Village, Ill.). Cutscum was from Fisher Scientific (Pittsburgh, Penn.), oleic acid, sodium taurocholate and sodium taurodeoxycholate were from Calbiochem (LaJolla, Calif.) and sodium boro[3H] hydride was from New England Nuclear (Boston, Mass.). Tissue Preparation Placenta obtained after delivery, and brain and liver specimens obtained at autopsy from controls and patients with Krabbe disease, were kept at -20'C until use. The tissues were homogenized in 4 volumes of 0.1% cutscum, 10 mM phosphate buffer, pH 6.0, in a Waring Blendor for three 1 min periods at top speed. The homogenates were centrifuged at 18,000 g for 30 min at 40C. After removal of cutscum using SM-2 beads [10], protein was determined by the Folin method [11] with bovine serum albumin as a standard. Fibroblasts from skin biopsies of controls and patients with Krabbe disease were cultivated, harvested, and lysed as previously described [9]. Enzyme Purification Galactosylceramide 3-galactosidase was purified from placenta by a four-step procedure as follows: (1) adsorption of placenta (500 g) supernatant on Sepharose-bound [12] concanavalin A (2 g Con A/200 ml Sepharose) in 0. 1% cutscum, 10 mM phosphate buffer, pH 6.0, and elution of the enzyme fraction with the same buffer containing 1 M a-methylglucoside and 0.5 M NaCl; (2) DEAE-cellulose (2.5 x 10 cm) chromatography with a linear gradient (500/500 ml) of NaCl, 0-0.4 M in 10 mM phosphate buffer, pH 6.0; (3) Bio Gel A 1.5 m (2.4 x 110 cm) gel filtration in 0.1 M phosphate buffer, pH 6.0; and (4) chromatography on Sepharose-bound [12] N-E-aminocaproyl-dihydrosphingosine (0.2 g ligand/20 ml Sepharose) [13] and elution with a linear gradient (50/50 ml) of ethylene glycol, 0%-50%. Electrophoresis on 5% or 7.5% polyacrylamide gel (75/1 acrylamide/bis acrylamide) was carried out at pH 7.0 with constant current of 8 mA/gel [14]. The gels were stained by 0.25% Coomassie Brilliant Blue G-250 in methanol/water/acetic acid (5/5/1). Enzyme Assays Activity of 13-glycosidases (at 37°C, pH 4.3) towards synthetic 4-methylumbelliferyl sub- strates (1 mM) was determined by an Aminco-Bowman spectrofluorometer with excitation at 365 nm and emission at 450 nm, after termination of the reaction with 0.1 M glycine-carbonate buffer, pH 9.8. Naphthol /8-galactosidase activity was demonstrated in precipitin lines as previously described [15]. Galactosylceramide and lactosylceramide were labeled with tritium by the galactose oxidase-sodium boro[3H]hydride method [16]. Activities of galactosyl- ceramidase and lactosylceramidase I were assayed according to Wenger et al. [17] and lactosylceramidase II according to Tanaka and Suzuki [6] with taurodeoxycholate [18] instead of the crude taurocholate. Radioactivity was measured in liquid scintillation spectrometer. Immunochemical Procedures Immunization of rabbits, immunoelectrophoresis on agarose gel and immunotitration of enzymic activities were carried out as previously described [15]. IgG fractions were prepared from antisera to galactosylceramide ,8-galactosidase and GM, f3-galactosidase [15] according to Levy and Sober [19], and the antibody concentration was determined by a quantitative precipitin test. Double immunodiffusion was performed in 0.8% agarose gel in phosphate buffered saline (PBS), 0.14 M NaCl 10 mM phosphate buffer pH 7.0. Single radial immunodiffusion was performed as previously described [9] except for the use of an IgG fraction of the antiserum as the first antibody and a goat antiserum to rabbit IgG as the second antibody. 646 BEN-YOSEPH ET AL.

RESULTS Enzyme Purit' The purification of galactosylceramide /3-galactosidase from 500 g placenta is summarized in table 1. The overall purification was 3,900-fold with a yield of 7%. The enzyme preparation was highly purified as shown by electrophoresis on SDS- polyacrylamide gel (fig. 1, left). The molecular weight is estimated to be around 120,000 and possibly represents identical size subunits of 700,000-800,000 oligomer. The purified enzyme was unstable and lost activity during storage at 40C or when subjected to freezing and thawing. Therefore, the purification procedure was carried out within 5 days, using only freshly prepared enzyme for characterization studies. Specific activities (nmol/h/mg protein) of the purified enzyme were as follows: galacto- sylceramide /3-galactosidase, 2,070; lactosylceramidase I, 5,180; lactosylceramidase II, 32.5; and 4-methylumbelliferyl /3-galactosidase, 760. No activity could be detected towards synthetic f-glucoside of N-acetyl-/3-glucosaminide. Antiserum Specificity The IgG fraction of the antigalactosylceramide /3-galactosidase antiserum was used to precipitate the enzyme from crude tissue homogenates. The various activities of ,3-galactosidase as well as /3-glucosidase and /3-N-acetylhexosaminidase activities towards 4-methylumbelliferyl substrates were assayed in the supernatants. The only activities precipitated from liver homogenate were those of galactosylceramidase and lactosylceramidase I (fig. 2). The antiserum IgG fraction gave a single precipitin line when examined against crude tissue preparations by both double gel immunodiffusion (fig. 3) and immunoelectrophoresis (fig. 1). No cross reactivity was observed with GMi ganglioside /3-galactosidase [15] or neutral /8-galactosidase [20]. Enzyme Antigenicit' Competition of the mutant enzyme with the normal was observed when immunoti- tration was performed on a mixture (1:1) of liver supernatants from a normal control and a patient with Krabbe disease (fig. 2). Precipitin lines of identity were demon- strated by double gel immunodiffusion between the purified placenta enzyme and crude preparations of brain, liver, and skin fibroblasts from normal controls and patients with

TABLE I PURIFICATION OF GALACTOSYLCERAMIDE f3-GALACTOSIDASE FROM PLACENTA

Specific Protein Activity Activity Purification Yield Step mg (nmoUh) (nmol/h/mg) -fold (%)

Placenta supernatant ...... 39,500 21,000 0.53 1 100. Concanavalin A adsorption ...... 208 11,400 54.8 103 54.3 DEAE-cellulose chromatography ...... 66.8 7,950 119 225 37.9 Bio-Gel A 1.5 M gel filtration ...... 16.1 5,230 325 613 24.9 chromatography ...... 0.72 1,490 2,070 3,900 7.1 NATURE OF MUTATION IN KRABBE DISEASE 647

FIG. 1. -SDS-polyacrylamide gel electrophoresis (left, 5% and right, 7.5%) and agarose gel im- munoelectrophoresis (middle) of purified (P) and crude (C) preparations of placenta galactosylceramide ,8-galactosidase using IgG fractions of antisera to placenta galactosylceramide f3-galactosidase (aK) and to liver GMI ganglioside ,f-galactosidase (aG). 648 BEN-YOSEPH ET AL. Krabbe disease (fig. 3, right). Similar electrophoretic mobility for both the normal and the mutant galactosylceramide /3-galactosidase was demonstrated by immunoelec- trophoresis using the antibodies to the normal enzyme. The precipitin lines obtained with tissue preparations from normal controls and patients with Niemann-Pick disease type A and GM, gangliosidosis type 1 revealed residual enzymic activity towards naphthol AS-BI-,8-galactoside. In contrast, almost no activity was visualized in the precipitin lines of patients with Krabbe disease (fig. 3, left).

Quantitation of CRM Calibration of the single radial immunodiffusion assay for the detection of an- tigenically cross reacting material of galactosylceramide f3-galactosidase is shown in figure 4. Known concentrations of the purified placenta enzyme were applied to agarose gel containing the IgG fraction of the anti-enzyme antiserum. The precipitin rings were visualized using a second antibody and stained for either protein or residual enzymic activity. Fibroblast lysates of four normal controls, four diseased controls, and four patients with Krabbe disease were assayed for 83-galactosidase activity towards galactosyl- ceramide. The CRM was quantitated in these cell preparations by the single radial immunodiffusion assay and the ratio of enzymic activity to CRM was calculated. The results are shown in table 2. Normal amount of CRM and normal specific activity of CRM were found in cell preparations from Niemann-Pick disease and GM,

00

50-

25-

.. . . O . 0.1 0.2 0.3 0.4 0.5 Antibody Added (mg) FIG. 2. -Immunotitration of galactosylceramide 18-galactosidase (0) and lactosylceramidase I (0) activities from 2 ml normal liver supernatant and galactosylceramide S-galactosidase (A) activity from 2 ml liver supernatants mixture (1:1) of normal control and Krabbe patient, by IgG fraction of antiserum to placenta galactosylceramide ,3-galactosidase (1.85 mg antibody/ml). NATURE OF MUTATION IN KRABBE DISEASE 649

iC:-..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... FIG. 3. -Double gel immunodiffusion of IgG fraction of antiserum to placenta galactosylceramide ,8-galactosidase (central well) against the purified enzyme (E) and crude homogenates of normal brain (B), Krabbe brain (b), normal liver (L), normal fibroblasts (F), and Krabbe fibroblasts (f). Left, stained for residual enzymic activity with naphthol-AS-BI-,/-galactoside; right, duplicate plate, unstained.

gangliosidosis. Both CRM and galactosylceramide ,3-galactosidase activity were lower than normal in fibroblasts from patients with I-cell disease, and this resulted in CRM specific activity within the normal range. However, in the fibroblasts of the Krabbe

80

60

E

40 -

CP

20

2 4 6 8 10 Galoctosylceramide 8-Golactosidase (;pg/ml) FIG. 4. -Single radial immunodiffusion of placenta galactosylceramide f8-galactosidase in 0.8% agarose gels in PBS containing IgG fraction of the anti-enzyme antiserum, 0.114 rug IgG/ml (0) and 0.285 mg IgG/ml (0). The precipitin rings were visualized by staining for activity (naphthol-AS-BI-/3-galactosidase) and protein respectively. 650 BEN-YOSEPH ET AL. TABLE 2 GALACTOSYLCERAMIDE ,8-GALACTOSIDASE ACTIVITY AND ANTIGENICALLY CROSS REACTING MATERIAL (CRM) IN CULTURED SKIN FIBROBLASTS

Cell Strain No. Activity* CRMt Activity/CRMf Normal ...... 4 3.8-6.9 0.41-0.65 8.7-10.6 Niemann-Pick disease ...... 1 5.7 0.55 10.4 GM, gangliosidosis ...... 1 6.4 0.61 10.5 I-cell disease ...... 2 0.9-1.0 0.08-0.10 10.0-11.2 Krabbe disease ...... 4 0.2-0.4 0.50-0.86 0.3- 0.5

* Galactosylceramide /3-galactosidase activity: nmollblmg protein. t CRM: Ag/mg protein. f CRM specific activity: nmol/h//ig CRM. patients, the very low activity was accompanied by normal to elevated amounts of CRM and the CRM specific activity was 1/20 to 1/30 of the normal. Quantitation of the CRM was also performed in one brain and two liver preparations from patients with Krabbe disease and found to be within the range of normal brain and liver controls.

DISCUSSION The antibody preparation used in this study seems to be monospecific to galactosyl- ceramide /3-galactosidase. In addition to a single precipitin line on double gel immunodiffusion (fig. 3) and immunoelectrophoresis (fig. 1) of crude tissue prepara- tions, we also found specific immunotitration of /3-galactosidase activities towards galactosylceramide and lactosylceramide in assay I conditions (fig. 2). Some overlap- ping specificities between GM, ganglioside f3-galactosidase and galactosylceramide ,/-galactosidase were observed by Tanaka and Suzuki [6, 21, 22], but the galactosyl- ceramide substrate showed almost exclusive specificity to the latter enzyme. The assay system for lactosylceramide I activity, although suboptimal for the galactosylceramide ,3-galactosidase enzyme, was capable of excluding the lactosylceramidase II activity of the GM, /8-galactosidase enzyme. The inability of our antiserum to cross react with GM1 ganglioside /8-galactosidase and neutral /3-galactosidase supports the idea that there are probably three different ,3-galactosidase enzymes. This is in agreement with the finding of Norden et al. [8] that neutral /8-galactosidase activity and /3-galactosidase activities towards galactosylceramide and lactosylceramide (I) were not precipitated by anti GM, ganglioside /3-galactosidase antiserum. In previous studies, we have similarly shown that insoluble conjugate of antibody to GM, /3-galactosidase did not adsorb lactosyl- ceramidase I activity [9], and antibody to neutral /3-galactosidase did not precipitate the activities of the two other ,3-galactosidase enzymes [20]. The specificity of the antiserum to galactosylceramide ,3-galactosidase enabled us to detect cross reacting material of this enzyme in the tissues of patients with Krabbe disease. Furthermore, the antigenic identity demonstrated between normal and mutant enzymes of various tissues by double gel immunodiffusion (fig. 3) and by competition in immunotitration (fig. 2), made possible the quantitation of catalytically deficient CRM with antibodies to the normal enzyme. The modified single radial immunodiffu- NATURE OF MUTATION IN KRABBE DISEASE 651 sion assay [9] is highly sensitive and allows the quantitation in tissue preparations containing 1-10Iug CRM per ml (fig. 4). The finding of normal to elevated amounts of CRM in Krabbe disease (table 2) indicates that the patients' tissues are probably synthesizing normal quantities of catalytically deficient, mutant enzyme. Thus the mutation in Krabbe disease appear to be structural and not regulatory; the defective gene produces altered galactosylceramide f3-galactosidase which lacks most of its enzymic activity but has retained most, if not all, of its antigenic determinants. SUMMARY Galactosylceramide /8-galactosidase cross reacting material was demonstrated in brain, liver, and skin fibroblasts from patients with Krabbe disease. The mutant enzyme was antigenically identical to the normal enzyme and exhibited similar electrophoretic mobility. Normal quantities of the catalytically deficient enzyme were measured in the patients' tissues by a sensitive single radial immunodiffusion assay, indicating that the mutation is in the structural gene for the enzyme protein.

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