Tohoku J. exp. Med., 1974, 112, 373-380

Hyperglycopeptiduria in Genetic Mucolipidoses

TADAO ORII, TAKAMICHI CHIBA, RYOJI MINAMI, KAZUKO S UKEGAWA and TooRu NAKAO Department of Pediatrics, Sapporo Medical College, Sapporo

ORII, T., CHmA, T., MINAMI, R., SUKEUAWA,K. and NAKAO, T. Hyper glycopeptiduria in Genetic Mucolipidoses. Tohoku J. exp. Med., 1974, 112 (4), 373-380 -Urinary cetylpyridinium chloride (CPC)-precipitates and non-CPC- precipitates in normal male children and seven patients with a new type of , GM1- type 1, I-cell disease, Hurler syndrome, Morquio syndrome, Gaucher's disease adult type and Tay-Sachs disease were studied using several methods including Sephadex G-25 gel filtration, ECTEOLA-cellulose column chromatography and enzymatic digestion with chondroitinase ABC. 1) Considerable amounts of glycopeptide fractions were detected in the urine of the patients with a new type of mucolipidosis, Gm1-gangliosidosis type 1, I-cell disease and also Gaucher's disease adult type compared with that of normal male children and other patients. 2) The total acid mucopolysaccharides excreted into the urine from two patients with Hurler syndrome and Morquio syndrome were much higher than those excreted in normal male children and other patients. 3) Large amounts of the chondroitinase ABC-resistant acid mucopolysaccharides were found in the urine of patients with Hurler syndrome, Morquio syndrome and Gm,-gangliosidosis type 1. mucolipidoses; glycopeptiduria; Gaucher's disease

A group of storage disease which exhibits signs and symptoms of both mucopolysaccharidoses and has tentatively been classified as the mucolipidoses by Spranger and Wiedemann (1970). With the exception of the Austin type of sulfatidosis, it has been reported by several workers that the patients with mucolipidosis generally show normal urinary excretion of uronic acid-containing mucopolysaccharides. However, in one patient with GMrganglio sidosis type 2, Wolfe et al. (1970) have found greatly elevated amount of urinary undersulfated keratan sulfate-like glycosaminoglycans. For these reasons, the present study was undertaken in order to determine whether large amounts of glycopeptides are excreted in some patients with mucolipidosis compared with those of mucopolysaccharidosis and sphingolipidosis.

MATERIALS AND METHODS

Analytical procedures were the same as previously described by Orii et al. (1972). Urine was collected from 75 healthy children (3?5 years) and from seven patients: each one case of a new type mucolipidosis (Orii et al. 1972), Gm,-gangliosidosis type 1, I-cell disease, Hurler syndrome, Morquio syndrome, Gaucher's disease adult type and Tay-Sachs

Received for publication, February 19, 1974. A part of this study was presented at the symposium of carbohydrate complex in the 44th Annual Meeting of the Japanese Biochemical Society, Oct., 1971, Sendai. 373 374 T. Orii et al.

disease. Urine specimens were kept frozen until assay.

Isolation of CPC-precipitates - Urine specimens were centrifuged at 1,200•~g for 10 min and the supernatants were diluted with distilled water and then specific gravity was adjusted to about 1,010. Further, urine samples were adjusted to pH 5.0 with 1.0 M HC1,

and a warm solution of 5% CPC was added (1.7 ml/100 ml of urine). After standing overnight at 4•Ž and centrifuging, precipitates were washed several times with 95% ethanol saturated with sodium acetate. The residues, containing the sodium salt of the crude mucopolysaccharides, were then dissolved in 10% sodium acetate and insoluble materials were removed by centrifugation. To the supernatant solution was added 4 volumes of ethanol, and the precipitates formed were washed several times with 80% ethanol. Furthermore, in order to determine relative proportions of chondroitinase ABC-

digested and -undigested mucopolysaccharides, the method 1 of Saito et al. (1968) was employed. The reaction mixtures contained, in 50 ƒÊl, 20 ,al of CPC-precipitate (contain- ing 80ƒÊg as uronic acid), 10 ƒÊl of Tris buffer, and chondroitinase ABC 10 ƒÊl (0.3 unit). These mixtures were incubated at 37•Ž for 3 hours and then the mixture, before and after the incubation, was spotted on a paper (Toyo Roshi No. 51A). After desalting by chromatography in n-butanol-ethanol-water (52:32:16 by vol.) for 48 hours, descending

paper chromatography was performed in n-butyric acid-0.5M ammonia (5:3, by vol.) for 36 hours. Each region of origin was cut out and mucopolysaccharides were eluted with 0.01 M HCl solution.

Isolation of non-CPC-precipitates -After removal of CPC-precipitates, the supernat ants were concentrated and dialyzed with Diaflo membrane UM-2 (Amicon, model 202),

precipitated by addition of 99% ethanol saturated with sodium acetate; the precipitates were washed with 95% ethanol saturated with sodium acetate dissolved in 10% sodium acetate and precipitated with 80% ethanol. Subsequently, crude non-CPC-precipitates were filtered through a column (1.8•~72.5 cm) of Sephadex G-25 (fine) with 180 ml of 0.05 M NaCI. 6-ml fractions were collected. Fractions filtered up to the void volume of the column from normal children and patients were collected, concentrated separately and applied to a column (1.8•~80 cm) of ECTEOLA-cellulose which was eluted with 600 ml each of water, 0.02 M HC1, 1.3 M NaCl and finally 4.0 M NaCl (Anseth et al. 1970). Each fraction was desalted on Sephadex G-25 column and lyophylized. A number of analytical determinations were carried out: uronic acid (Dische 1947), hexosamine (Svennerholm 1956), hexose (Roe 1955), sialic acid (Warren 1959), total sulfate (Dodgson and Price 1962), protein (Lowry et al. 1951), creatinine (Bonsnes and Taussky 1945), and paper chromatographic identification of hexose and hexosamine (Strecker and Montreuil 1971; Trevelyan et al. 1950).

RESULTS

Tables 1 and 2 indicate that two cases of Hurler and Morquio syndromes excrete large amounts of the urinary acid mucopolysaccharides compared with those of normal male children and other patients, and Table 1 also shows that with the exception of two cases of Hurler syndrome and Tay-Sachs disease, values of the dry weight of non-CPC-precipitates from all of the patients are much higher than that from normal male children. Further, the composition of non- CPC-precipitates derived from the urine of seven patients is shown in Table 3. Considerable amounts of non-CPC-precipitates were obtained in the urine of four patients, Cases 1, 2, 6 and 7. These non-CPC-precipitates contained large amounts of hexose or hexosamine and little uronic acid. Table 4 indicates that three cases of Hurler, Morquio syndromes and also GM1-gangliosidosis have very Genetic Mucolipidoses 375

TABLE1. Excretion of urinary crude acid mucopolysaccharideand glycopeptide fractions in seven patients with genetic storage disease

* Percent of total dry weight is given in parentheses .

TABLE 2. Composition of crude acid mucopolysaccharide fraction from the urine

TABLE 3. Composition of crude non-cetylpyridinium chloride precipitable glycopeptidefractions from the urine

high proportions of the chondroitinase ABC-resistant mucopolysaccharidesthan those from other patients and normal male children. Gel filtration of the non- CPC-precipitateson Sephadex G-25 column showed that each fraction filtered up to the void volume contained 80.2%, 48.0%, 40.2%, 90.4%, 66.8%, 58.6%, 69.7% and 74.4% of uronic acid in Cases 1?7 and normal male children, respectively. 376 T. Orii et al.

TABLE4. Analysis of urinary acid mucopolysaccharidesafter chondroitinase ABC digestion

* Mole ratios based on hexosamine=1 .0.

TABLE 5. ECTEOLA -cellulose column fractionation of non-cetylpyridinium precipitable glycopeptide fractions filtered up to the void volume through a column of Sephadex G-25 ,fine

Percent of total dry weight is given in parentheses.

Each fraction filtered up to the void volume on Sephadex G-25 gel filtration, was applied to a column of ECTEOLA-cellulose. Table 5 indicates that 86.3% of the total dry weight in mg per g of creatinine from normal male children and 77.6% -89.3% of those from seven patients were eluted up to a HCl concentration of 0.02 M. Table 6 also shows that 97.1% of the total hexosamine from normal male children and 74.0% -92.8% of those from seven patients were eluted up to a HCl concentration of 0.02 M. These main fractions were poor in uronic acid and sulfate and rich in hexose and hexosamine. Significantamounts of these glycopep tide fractions were obtained from four patients with a new type of mucolipidosis, Genetic Mucolipidoses 377

TABLE6. Compositions of non-cetylpyridinium chloride precipitable glycopeptide fractions after ECTEOLA-cellulose column fractionation

GM1-gangliosidosis, I-cell disease and Gaucher's disease. Table 7 indicates that H2O fractions from the normal male children and seven patients after the ECTEOLA- cellulose column chromatography showed on analysis high molar ratios of hexose to hexosamine and low molar ratios of sulfate or uronic acid to hexosamine, respectively, and composition of the fractions eluted up to the concentration of 0.02 M HCl did not differ significantly between them. It appears in Fig. 1 that the predominant carbohydrate constituents in the combined H2O and 0.02 M HC1 fractions derived from Cases 1, 2, 3 and normal male children are galactose and glucosamine. 378 T. Orii et al.

TABLE7. Mole ratios of non-cetylpyridinium chloride precipitable glycopeptidefractions

* Mole ratios based on hexosamine=1 .00.

DISCUSSION With the exception of one patient with GM,-gangliosidosis type 2, pattern of CPC-precipitates and non-CPC-precipitates in mucolipidoses has been scarcely reported. In one patient with fucosidosis, Durand et al. (1966) reported that excess amount of an acid mucoid, unidentified, abnormal material which was not acid mucopolysaccharides was present in urinary sediment. Further, in two patients with mucolipidosis type 1, Spranger et al. (1968) detected increased amounts of hexosamine-containing uromucoid. However, these abnormal materi als excreted have not been studied in detail. In the present investigation, it is of interest that large amounts of glycopeptides are excreted into the urine from three cases with mucolipidosis differing from those of mucopolysaccharidosis or sphingolipidosis. Furthermore, the data obtained indicate an increase of urinary Genetic Mucolipidoses 379

Fig. 1. Paper chromatogram of the combined H2O and 0.02 M HCl fractions from ECTEOLA-cellulose chromatography after hydration with 1.0 M HCl. 1, standard; 2, normal children; 3, I-cell disease; 4, new type of mucolipidosis; 5, GMl-gangliosidosis type 1; 6, normal children; 7, standard; a, glucose; b , galac tose; c, glucosamine; d, galactosamine.

glycopeptide excretion in one patient with Gaucher's disease of adult type. In this disease, therefore, it seems possible that not only , but also glycoprotein accumulate excessively in the viscera. Recently, an excellent study on an increase of mannose-containing trisaccharide in urine of three patients with mannosidosis was reported by Norden et al. (1973). Thus, it seems highly probable that as indicated by the present study, Wolfe et al. (1970), and Norden et al. (1973) the glycopeptide and its heterogeneous inter- mediates in the sequence of glycoprotein catabolism are excessively excreted in the urine of mucolipidoses. Therefore, these studies may give additional informa tion about the pathogenesis and detection of various mucolipidoses.

References

1) Anseth, A., Antonopoulos, C.A., Bjelle, A. & Fransson, L-A. (1970) Fractionation and quantitative determination of keratan sulfate using cetylpyridinium chloride and ECTEOLA-cellulose. Biochim. Biophys. Acta, 215, 522-526. 2) Bonsnes, R.W. & Taussky, H.H. (1945) On the colorimetric determination of creatinine by the Jaffe reaction. J. biol. Chem., 158, 581-591. 3) Dische, Z. (1947) A new specific color reaction of hexuronic acids. J. biol. Chem., 167, 189-198. 4) Dodgson, K.S. & Price, R.G. (1962) A note on the determination of the ester sulphate content of sulphated polysaccharides. Biochem. J., 84, 106-110. 380 T. Orii et al.

5) Durand, P., Borrone, C. & Della Cella, G. (1966) A new mucopolysaccharide lipid storage disease? Lancet 2, 1313-1314. 6) Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measure ment with the folin phenol reagent. J. biol. Chem., 193, 265-275. 7) Norden, N.E., Lundblad, A., Svensson, S., Ockerman, P.-A. & Autio, S. (1973) A mannose-containing trisaccharide isolated from urines of three patients with manno sidosis. J. biol. Chem. 248, 6210-6215. 8) Orii, T., Minami, R., Sukegawa, K., Sato, S., Tsugawa, S., Horino, K., Miura, R. & Nakao, T. (1972) A new type of mucolipidosis with ƒÀ-galactosidase deficiency and

glycopeptiduria. Tohoku J. exp. Med., 107, 303-315. 9) Roe, J.H. (1955) The determination of sugar in blood and spinal fluid with anthrone reagent. J. biol. Chem., 212, 335-343. 10) Saito, H., Yamagata, T. & Suzuki, S. (1968) Enzymatic methods for the determina tion of small quantities of isomeric chondroitin sulfates. J. biol. Chem., 243, 1536- 1542. 11) Spranger, J.W. & Wiedemann, H.-R. (1970) The genetic mucolipidoses. Humangenetik, 9, 113-139. 12) Spranger, J., Wiedemann, H.-R., Tolksdorf, M., Graucob, E. & Caesar, R. (1968) Lipomucopolysaccharidose. Z. Kinderheilk., 103, 285-306. 13) Strecker, G. & Montreuil, J. (1971) Description d'une oligosaccharidosurie accompag nant une gangliosidose GM2 h deficit total en N-acetyl-. Clin. Chim. Acta, 33, 395-401. 14) Svennerholm, L. (1956) The determination of hexosamines with special reference to nervous tissue. Acta Soc. Med. upsalien., 61, 287-306.

15) Trevelyan, W.E., Procter, D.P. & Harrison, J.S. (1950) Detection of sugars on paper chromatograms. Nature (Lond.), 166, 444-445. 16) Warren, L. (1959) The thiobarbituric acid assay of sialic acids. J. biol. Chem., 234,

1971-1975. 17) Wolfe, L.S., Callahan, J., Fawcett, J.S., Andermann, F. & Scriver, C.R. (1970) GM1-

gangliosidosis without chondrodystrophy or visceromegaly. Neurology (Minneap.), 20, 23-44.