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The Nature of Mutation in Krabbe Disease

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The Nature of Mutation in Krabbe Disease Am J Hum Genet 30:644-652, 1978 The Nature of Mutation in Krabbe Disease YOAV BEN-YOSEPH,1 MELINDA HUNGERFORD, AND HENRY L. NADLER Krabbe disease (globoid cell leukodystrophy) 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 galactosylceramide /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- ceramide under different assay conditions [6] and /3-galactosidase activities towards GM, ganglioside, 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 Sphingosine chromatography ................. 0.72 1,490
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