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AB Variant of Infantile GM2 Gangliosidosis: Deficiency of A

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AB Variant of Infantile GM2 Gangliosidosis: Deficiency of A Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 3979-3983, August 1978 Medical Sciences AB variant of infantile GM2 gangliosidosis: Deficiency of a factor necessary for stimulation of hexosaminidase A-catalyzed degradation of ganglioside GM2 and glycolipid GA2 (lipid storage disease/activator deficiency) E. CONZELMANN AND K. SANDHOFF Max-Planck-Institut fur Psychiatrie, Neurochemische Abteilung, Kraepelinstrasse 2, D-8000 Munchen 40, Federal Republic of Germany Communicated by Saul Roseman, May 24, 1978 ABSTRACT Human kidney extracts heated to 600 and de- the degradation of gangliosides (10, 12). This paper presents void of hexosaminidase activity (2-acetamido-2-deoxy-13-Dglu- evidence that a component of an activating factor necessary for coside acetamidodeoxyglucohydrolase EC 3.2.1.30) stimulate the degradation of ganglioside GM2 and glycolipid GA2 cata- more than 20-fold the hexosaminidase A-catalyzed degradation of ganglioside GM2 and of glycolipid GA2, the neuronal storage lyzed by hexosaminidase A is defective in variant AB of in- compounds of GM2 gangliosidosis. The stimulating factor of this fantile GM2 gangliosidosis. extract, which is labile at temperatures above 60°, is also present in kidney extracts from patients with infantile GM2 gangliosi- MATERIALS AND METHODS dosis having a deficiency of hexosaminidase A (Tay-Sachs disease, variant B) and a deficiency of hexosaminidases A and Materials. 4-Methylumbelliferyl-2-acetamido-2-deoxy- B (variant 0). Evidence is presented that this factor is defective ,B-D-glucopyranoside (MUFGlcNAc) and N-acetyl-D-galac- in the AB-variant of infantile GM2 gangliosidosis which is tosamine (GalNAc) were from Koch-Light (England); sodium characterized by an accumulation of glycolipids GM2 and GA taurodeoxycholate, trypsin, and trypsin inhibitor, from Sigma despite the fact that the degrading enzymes, hexosaminidases Chemical Company (St. Louis, MO); Pronase P (from Strep- A and B, retain normal activity levels. Thus, variant AB is an example of a fatal lipid storage disease that is caused not by a tomyces griseus), from Serva (Heidelberg). DEAE-cellulose defect of a degrading enzyme but rather by a defective factor (DE-52) was obtained from Whatman (Springfield Mill, Great necessary for the interaction of lipid substrates and the water- Britain), Bio-Gel P-200 from Bio-Rad Laboratories (Munich, soluble hydrolase. Federal Republic of Germany). Ganglioside GM2, tritiated in its GalNAc portion (2 ,uCi/,umol) (13), and glycolipid GA2 (190 Infantile GM2 gangliosidosis is a fatal inherited storage disease ,uCi/,umol), labeled by reduction of the double bond in the characterized by the accumulation of ganglioside GM2 and its sphingosine moiety, were prepared as described (6). All other asialo derivative, GA2, in nervous tissue (1). Biochemically, three reagents were analytically pure or of the best grade available. variants can be distinguished, two of which could hitherto be Organs from patients with variant 0, B, or AB of infantile GM2 explained in terms of enzyme defects: patients with variant B gangliosidosis and from normals were deep frozen within 6 hr ("Tay-Sachs disease") lack hexosaminidase A (2-acetamido- after death and kept for 2-3 yr at -700 until used. 2-deoxy-f-D-glucoside acetamidodeoxyglucohydrolase, EC Preparation of Tissue Extracts. Frozen human tissues (0.5-1 3.2.1.30) (2, 3), the enzyme responsible for the further degra- g) were thawed, homogenized in 4 vol of distilled water with dation of the ganglioside GM2 (4), whereas in patients with an Ultra-Turrax homogenizer (Janke & Kunkel, Staufen, variant 0, major hexosaminidase isoenzymes A and B both are Federal Republic of Germany), and centrifuged at 13,000 X missing (5, 6), presumably due to the defect of a common g for 10 min. To obtain a preparation of the activating factor subunit (7). For the variant AB, however, no such enzyme de- virtually free of hexosaminidases, the extract was heated at 600 ficiency could be demonstrated. Patients having this variant for 1 hr, centrifuged, and the precipitated protein was dis- retain normal or show elevated levels of both hexosaminidases carded. Protein was determined according to Lowry et al. (14) in all organs examined (3, 6), yet the ganglioside GM2 never- with crystalline bovine serum albumin as standard. theless accumulates in the brain tissue at the same rate as in the Ion Exchange Chromatography. Heated tissue extract (1.2 B and 0 variants, and the clinical symptoms and course of the g of protein in 230 ml) prepared as described above was di- disease are the same as in these variants. Biochemical and im- alyzed against 10 mM phosphate buffer (pH 6.5) and loaded munological comparison of an AB patient's hexosaminidase A onto a DEAE-cellulose column (Whatman DE-52; volume, 300 with the enzyme from normal subjects revealed no difference ml) that had been equilibrated with the same buffer. After a (8). In particular, the patient's storage compounds, ganglioside washing with 2 vol of buffer, the column was eluted with a GM2 and glycolipid GA2, were split by the patient's hexosami- nidase A in the presence of detergent at a normal rate, thus linear NaCl gradient, 0-0.5 M in 1 liter of 10 mM phosphate practically ruling out the possibility of a mutant hexosaminidase buffer (pH 6.5). Fractions (25 ml) were collected and assayed A. for protein and stimulating factor as described below. Recent publications have demonstrated the necessity for the Proteolytic Digestion. Components a and f (50,l each) presence of a nonenzymic protein to bring about the interaction obtained after gel filtration of the stimulating factor were in- of some water-soluble hydrolases (9-11) with their sphingolipid substrates. Abbreviations: MUFGIcNAc, 4-methylumbelliferyl-2-acetamido-2- Such activator proteins have also been reported for deoxy-fl-D-glucopyranoside; GaINAc, N-acetyl-D-galactosamine; NeuAc, N-acetylneuraminic acid; Cer, ceramide; glycosphingolipid The costs of publication of this article were defrayed in part by the GA2, gangliotriaosylceramide (GgOse3Cer; GalNAc31-4Gal- payment of page charges. This article must therefore be hereby marked f1-4GlI31-1Cer); ganglioside GM2, II3NeuAc-GgOse3Cer (GalN- "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Acf11-4Gal(3. -2aNeuAc)31-4Glfl-BCer); ganglioside GM3, this fact. NeuAca2-3Gal31-4Glcll-lCer. 3979 Downloaded by guest on September 29, 2021 3980 Medical Sciences: Conzelmann and Sandhoff Proc. Natl. Acad. Sci. USA 75 (1978) cubated with Pronase P (0.5 mg) at 370 (pH 6.5, 10 hr). Under in all three. The solvent systems were: (i) chloroform/metha- these conditions, both components were inactivated by more nol/water, 14:6:1 (vol/vol); (if) chloroform/methanol/con- than 90%. Controls were run under the same conditions except centrated NH4OH, 65:35:4 (vol/vol); and (hi) chloroform/ that the protease was added only after incubation to show that methanol/0.25% NaCl in water, 62.38:8 (vol/vol). Lipids were the subsequent assay for the stimulating components is not visualized with acetic acid/sulfuric acid/anisaldehyde, 50:1:0.2 disturbed by the protease which is inactive at acidic pH. (vol/vol) 15 min at 1400. The distribution of Preparation of Purified Hexosaminidase A. Hexosamin- radioactivity on idase A was isolated from normal human liver as described (4). the plates was measured with a , camera LB 290B (Berthold, Bovine serum albumin (0.1 mg/ml) was added and the enzyme Wildbad, Federal Republic of Germany). solution was stored at -70°. Enzyme Assays. Hexosaminidase activity was tested as de- RESULTS scribed (4), with MUFGlcNAc as substrate; 1 unit of hexo- Stimulation of Enzymic Hydrolysis of Glycolipids by saminidase activity releases 1 1mol of 4-methylumbelliferone Heated Extracts of Organs. Tissue extracts whose hexosami- per min at 37°. nidase activity had been destroyed by heating to 600 for 1 hr Ganglioside Gm2 N-acetyl-ft-D-galactosaminidase was es- stimulated the degradation of ganglioside Gm catalyzed by sentially determined according to O'Brien et al. (13). Gangli- purified hexosaminidase A (Fig. 1). Kidney extracts activated oside GM2 (10 nmol), labeled in its N-acetyl-D-galactosamine the enzymic hydrolysis of ganghoside more than 18-fold lin- portion (2 ACi/timol) (12), in chloroform/methanol, 2:1 (vol/ early with the amount of extract protein added, whereas ex- vol), was pipetted into the reaction vessel and dried under a tracts fromh brain and liver showed only a slight stream of nitrogen. Citrate buffer (pH 4.2; 4 Mmol) and water activation at were added and the mixture was sonicated for 5 sec. As indi- low protein concentrations (3- to 4-fold) and this diminished cated in the legends of figures, extract, supernatant of heat- with higher concentrations of protein. Corresponding obser- inactivated extract, or detergent and purified hexosaminidase vations were obtained for the enzymic degradation of glycolipid were added to give a final volume of 40 Al. The mixture-was GsA. Because kidney samples were available from variant AB vigorously shaken and incubated at 370 for 2-4 hr. The reaction as well as from other variants of infantile GM2 gangliosidosis, was stopped by the addition of 750 ll of CHC13/CH30H/2.5 further investigations on this activating factor were carried out M aqueous NH&, 80:201 (vol/vol), followed by 0.5 ml of 1 mM with these different kidney extracts. The activating factor was N-acetyl-D-galactosamine in water. After thorough shaking, stable up to 600 but labile at higher temperatures (Figs. 2 and the phases were separated by centrifugation. The upper phase 3). Because of this stability at 600, it is possible to prepare kidney was collected and loaded onto a small column (1 ml) of extracts rich in activating factor but essentially free of hexo- DEAE-cellulose that had previously been washed with distilled saminidase activity, so that these extracts degrade neither water.
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