Gargoylism: hydrolysis of β-galactosides and tissue accumulation of galactose- and mannose-containing compounds Björn Hultberg, … , Per-Arne Öckerman, Arne Dahlqvist J Clin Invest. 1970;49(2):216-224. https://doi.org/10.1172/JCI106230. Research Article The sugars present in hydrolyzed extracts of human liver and brain were analyzed by gasliquid chromatography after conversion to their alditol acetates. The samples analyzed were obtained from control subjects, patients with gargoylism, and patients with a few other kinds of storage disorders. Accumulation of galactose was demonstrated in the liver and the brain of two patients with gargoylism, and in the liver samples, high levels of mannose were found too. We also studied the hydrolysis of a number of galactosides by homogenates from different tissues in the control subjects and in the patients. Separation methods and kinetic studies demonstrated the presence in normal human tissues of two different β- galactosidases, which we call enzyme A and enzyme B, respectively. Enzyme A hydrolyzed all the β-galactosides tested. Enzyme B hydrolyzed the synthetic substrates tested (4-methylumbelliferyl-, p-nitrophenyl-, o-nitrophenyl-, and phenyl-β- galactoside) but not the natural substrates tested (ceramide-β-galactoside, ceramide lactoside, transferrin glycopeptide, and keratan sulfate). Enzyme B also exerted β-glucosidase activity. In various tissues from patients with gargoylism, deficiency of β-galactosidase A could be demonstrated. It is suggested that the high level of galactose found in the hydrolyzed extracts of tissues from gargoylism patients is due to storage of galactose-rich glycosaminoglycans and glycopeptides, and that this storage is a result of the deficiency of β- galactosidase A. The high level of mannose in the […] Find the latest version: https://jci.me/106230/pdf Gargoylism: Hydrolysis of 0 - Galactosides and Tissue Accumulation of Galactose- and Mannose-Containing Compounds Bj6RN HULTBERG, PER-ARNE OCKERMAN, and Apm DAHLQVIST From the Laboratory of Clinical Chemistry and the Research Department, University Hospital, S-220 05 Lund, Sweden A B S T R A C T The sugars present in hydrolyzed ex- INTRODUCTION tracts of human liver and brain were analyzed by gas- In gargoylism the metabolism of glycosaminoglycans liquid chromatography after conversion to their alditol (earlier called acid mucopolysaccharides) and glyco- acetates. The samples analyzed were obtained from lipids (gangliosides) is disturbed (1, 2). The clinical control subjects, patients with gargoylism, and patients symptoms and signs of the pathological accumulation with a few other kinds of storage disorders. Accumula- of different substances involve many organs, are pro- tion of galactose was demonstrated in the liver and the gressive, and finally lead to death. brain of two patients with gargoylism, and in the liver On the basis of electronmicroscopical studies, gargoyl- samples, high levels of mannose were found too. We ism has been suggested to be a lysosomal disease (3). also studied the hydrolysis of a number of galactosides Recently, markedly decreased activity of MU-fi-galactosi- by homogenates from different tissues in the control sub- dase' (P-D-galactoside galactohydrolase, EC 3.2.1.23) jects and in the patients. Separation methods and kinetic was demonstrated (4-7). P-galactosidase activity is studies demonstrated the presence in normal human tis- known to be present in the lysosomes of man (6) as well sues of two different f-galactosidases, which we call en- as in those of the animal (8). zyme A and enzyme B, respectively. Enzyme A hydro- The apparent possibility exists that the demonstrated lyzed all the P-galactosides tested. Enzyme B hydrolyzed decrease in p-galactosidase activity in patients with the synthetic substrates tested (4-methylumbelliferyl-, gargoylism may reflect the absence of an enzyme which p-nitrophenyl-, o-nitrophenyl-, and phenyl-P-galactoside) is necessary for the normal catabolism of the glyco- but not the natural substrates tested (ceramide-i3-galac- saminoglycans and glycolipids which are known to be toside, ceramide lactoside, transferrin glycopeptide, and stored in pathological amounts in this disease. We do keratan sulfate). Enzyme B also exerted P-glucosidase not known whether these glycosaminoglycans and gly- activity. In various tissues from patients with gargoyl- *colipids contain much galactose, nor do we know which ism, deficiency of P-galactosidase A could be demon- is the natural substrate for the p-galactosidase which is strated. deficient in gargoylism. It is suggested that the high level of galactose found In the present study we have, therefore, measured the in the hydrolyzed extracts of tissues from gargoylism level of galactose and other sugars in hydrolyzed tissue patients is due to storage of galactose-rich glycosamino- extracts from patients with gargoylism, patients with glycans and glycopeptides, and that this storage is a other kinds of storage diseases, and normal control sub- result of the deficiency of P-galactosidase A. jects. We have also tried to characterize the i3-galactosi- The high level of mannose in the liver from gargoyl- dase deficiency by using a large number of substrates ism patients seems to indicate storage of glycopeptide, and by performing separation experiments and enzyme adding a new group of substances to those known to be kinetic studies with different kinds of tissues. The sub- stored in gargoylism. strates used include four synthetic P-galactosides (MU- Received for publication 2 June 1969 and in revised form 'Abbreviations: MU, 4-methylumbelliferyl; PNP, para- 2 July 1969. nitrophenyl; ONP, ortho-nitrophenyl; P, phenyl. 216 The Journal of Clinical Investigation Volume 49 1970 fB-galactoside, PNP-P-galactoside, ONP-fi-galactoside, A 1-2 ,l sample of the chloroform solution was injected and P-fi-galactoside), two galactolipids (ceramide-p- into the gas chromatograph (Aerograph, model 1520). The columns were 2 m long, made from * inch stainless steel, galactoside and ceramide-P-lactoside), one glycopeptide and were filled with a mixture containing 1.5% ethylene derived from transferrin, and one glycosaminoglycan glycol succinate and 1.5% silicone oil (XF-1150) on 100-120 (keratan sulfate). mesh Gas-chrom P, purchased from Professor G. Widmark, Department of Analytical Chemistry, University of Stock- METHODS holm. The standard separation conditions were: injector temperature 260'C, detector cell temperature 217'C, and Tissue preparations column temperature 1950C. Normal controls. Tissue specimens were obtained 3 hr When we wanted to obtain a good separation between the postmortem from two children killed in a road accident, and sugars appearing early in the chromatography, we used a 5-15 hr postmortem from adults who had died of various lower column temperature (temperature programming) diseases supposed not to engage liver, spleen, kidney, or during the first 5-10 min of the run. brain primarily. Secondary damage to these tissues could Commercial monosaccharides of purest grade available not be excluded, but more strictly normal tissues could were used as reference substances. A small amount of not be obtained. rhamnose was added to each tissue sample before analysis Gargoylism patients. Three of the patients with gargoyl- and served as an internal standard. The peak area for each ism have been described earlier (6) and will, in accord with component found was calculated by triangulation. The rela- their previous nomenclature, be named case 1 case 4, and tive detector response for each monosaccharide was de- case 7, respectively. Samples have now been obtained from termined. one further patient, a 7 yr old boy, who will be named To test the reliability of the assay with gas-liquid chro- case 8. These patients had clinical symptoms and signs in matography, the amount of glucose and galactose recovered all respects compatible with the diagnosis of gargoylism, and in tissue extracts was compared with the amounts found large amounts of dermatan sulfate and heparan sulfate were on enzymatic analysis (method described in reference 11, also found in the urine. The tissues from cases 4 and 7 were compare below). Similar results were obtained. frozen 7 hr and about 15 hr, respectively, postmortem. The tissues from cases 1 and 8 were obtained by surgical biopsy Enzyme activity determinations and were frozen immediately. Substrates. MU-fi-galactoside, ONP-j8-galactoside, PNP- Other storage disorders. Tissues from three patients with from the Gaucher's disease and one patient with juvenile amaurotic fi-galactoside, and P-p-galactoside were obtained idiocy were studied. They had been stored for up to 4 yr same sources as described previously (5, 12). at -20'C. Tissues from one patient with Tay-Sachs' dis- Ceramide-P-galactoside (N-lignoceroyl-DL-sphingosyl-fi-D- with a late-infantile storage disorder galactoside, kerasin) and ceramide-,8-lactoside (N-lignoceroyl- ease and two patients DL-sphingosyl-fi-D-lactoside, cytolipin H) were obtained from (progressive encephalopathy with storage of lipofuscin-like Seravac Laboratories, Maidenhead, England. Transferrin material in a pair of female twins, 13 yr old) had been glycopeptide (sialic acid-free) was prepared from transferrin stored for between a few weeks and a few months at - 20'C. (a gift from AB KABI, Stockholm) by treatment with pronase and mild acid hydrolysis as described by Jamieson Determination of sugars by gas-liquid (13). Keratan sulfate was a gift from Dr. L.-A. Fransson chromatography (Department of Physiological Chemistry, University of The tissue was extracted with lipid solvents. 10-40 mg dry, Lund) ( 14). lipid-free extract was used for hydrolysis. Rhamnose was Galactose dehydrogenase. This enzyme was obtained from added and the material was neutralized. All these procedures C. F. Boehringer & Sons, Mannheim, West Germany. were as described by Walborg, Christensson, and Gardell Performance of the assay. MU-f-galactosidase activity (9). was assayed with the same method as that described in For reduction and acetylation' the freeze-vacuum dried earlier publications (5, 6, 15, 16). Acetate buffer pH 4.5 was hydrolyzed extract was dissolved in 1 ml of water.