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Urinary excretion patterns of sialoglycoconjugates in patients with mucolipidosis
BIOCHEMICAL
MEDICINF
27, 2X-243
f lY%)
Urinary Excretion Patterns of Sialoglycoconjugates Mucolipidosis
in Patients with
MASAI KOSEKI,’ MICHIKO EJIRI, KAZUO EJIRI, SHIGERUINO, AND KOICHI TSURUMI* Fukushima Institute of Health and *Department oj.Biochemistry. College. Fukushimu 960. Japan
Fukushima Medical
Received September 8, 1981
In 1977 Spranger et al. (1) observed diminished activity of sialidase (EC 3.2.1.18) and excessive tissue accumulation as well as urinary excretion of sialoglycoconjugates (SGC) in one patient with mucolipidosis (ML) I. Accordingly they first proposed the term “sialidosis” for this disease. Later investigations, however, have demonstrated the presence of several variant forms of sialidase deficiency (2-8). The affected patients do not always show the clinical signs characteristic of ML I. The common clinical signs and biochemical findings are macular cherry-red spots, myoclonus, loss of visual acuity, and the disturbed metabolism of SGC. Recently Lowden and O’Brien (9) classified these diseases into two types on the basis of clinical findings, (type 1) normosomatic group and (type 2) dysmorphic group, the latter group being divided into two subgroups, juvenile onset and infantile onset. Although the deficiency of sialidase is the basic defect of these diseases, the biochemical basis for the different clinical types of sialidosis remains unclear. The results of a comparative study of the excretion patterns of sialyl-oligosaccharides in urine of patients with several types of ML are dealt with in this report. MATERIALS AND METHODS Urine specimens. Pooled urine (5000 ml) from a patient with ML I (Y. K., a lPyear-old boy) was supplied by Dr. R. Minami, Sapporo Medical College, Sapporo. The clinical and cytological studies on this ’ To whom correspondence should be addressed: Fukushima Institute of Health, 16-6 Mitouchi, Hokida, Fukushima 960. Japan. 232 0006-2944/82/020232-12$02.00/O Copyright All rights
8 1982 by Academic Press. Inc. of reproductmn in any form recerwd
URINARY SIALOGLYCOCONJUCATES
233
patient have been reported by Orii et al. (2). Urine specimens from 2 subjects with hypersialosiduria, myoclonus, and macular cherry-red spots (a 7-year-old girl, A. T., 78 ml, and her 12-year-old brother, K. T., 115 ml), were supplied by Professor T. Kitagawa and Dr. M. Owada, Nihon University School of Medicine, Tokyo. The clinical study on these patients has been reported by Owada et al. (IO). Urine specimens from 2 subjects with ML II (I-cell disease), a 6-year-old boy (T. N., 450 ml) and his 1l-year-old sister (N. N., 530 ml), were supplied by Professor T. Kitagawa and Dr. M. Owada. The clinical and biochemical studies on these patients have been reported by Sakiyama (11). A urine specimen (450 ml) from a patient with ML II (W. O., a l-year-old girl) was supplied by Dr. R. Minami. The clinical study on this patient has been reported by Takeuchi et al. (12). Normal human urine (16,000 ml) was collected from young healthy boys ranging from 10 to 11 years of age and was used for the isolation and fractionation of urinary SGC. Morning urine was collected from 10 healthy men and 8 healthy women and was used for the determination of normal excretion levels of bound sialic acid. All urine specimens were stored frozen until used. Analytical methods. Total sialic acid was determined by the orcinol method of Bohm et al. (13), and free sialic acid by the thiobarbituric acid method of Warren (14). The results were expressed as N-acetylneuraminic acid (NeuAc). Total neutral sugars were determined by the anthrone method (15) using a mixture of equal amounts of galactose and mannose as standard. The molar compositions of neutral sugars were determined by gas-liquid chromatography as their alditol trifluoroacetates. The samples for the assay were hydrolyzed with 2.5 N trifluoroacetic acid at 100°C for 5 hr. The dried hydrolyzates were reduced with sodium borohydride, trifluoroacetylated by the method of Tamura et al. (16) and analyzed by gas-liquid chromatography on a glass column (3 mm x 200 cm) packed with 3% XF-1105 on Gaschrom Q (SO-100 mesh). The column temperature was programmed from 115 to 145°C at a rate of l”C/min. Total hexosamines were determined by the Elson-Morgan reaction described by Blix (17), and the samples for the assay were hydrolyzed with 2 N hydrochloric acid at 100°C for 14 hr. Total protein was determined by the method of Lowry et al. (18) using human serum protein as standard. Uranic acid was determined by the carbazole method of Bitter and Muir (19) using glucuronic acid as standard. The compositions of amino acids and amino sugars were determined on a JLC-5AH automatic amino acid analyzer. The samples for the assay were hydrolyzed with 6 N hydrochloric acid at 110°C for 22 hr. Urinary SGC were isolated and determined by the method described by Koseki et al. (20). For the determination of glycosaminoglycans, the urine samples were diluted to obtain a specific gravity of 1.020 or less and adjusted to pH
34
KOSEKI
ET AL.
6.0. To 10 ml of these samples was added 0.5 ml of 5% cetylpyridinium chloride. After standing overnight at 4°C. the precipitates formed were separated, washed with NaGsaturated ethanol. and dissolved in 3 ml of 0.01 N sodium hydroxide. Uranic acid contents of these samples were determined by the carbazole method. Thin-layer chromatography of sialyl-oligosaccharides was performed according to the method of ohman (21), and spots of reducing sugars were visualized by spraying the aniline hydrogen phthalate reagent. EXPERIMENTS
AND RESULTS
Determination qf Urirtary Sialic Acid and Uranic Acid Urinary sialic acid in patients with ML and in healthy individuals was determined by the charcoal adsorption method described in our previous paper (20). Urinary glycosaminoglycans were precipitated with cetylpyridinium chloride, and uranic acid contents of the precipitates were determined by the carbazole method. The results are listed in Table I. As shown in the table, increased urinary excretion of sialic acid was observed in the patients with so-called sialidosis. The patients with Icell disease also excreted increased amounts of sialic acid, the excretion levels of which were indistinguishable from those of the patients with sialidosis. In contrast, the excretion levels of uranic acid were within TABLE URINARY
EXCRETION
OF SIALIC ACID
AND
I
URONK
ACID
IN PATIENTS
Syndrome
(years)
Sex
Sialic acid
Uranic acid“ 13.3 (IA- 2.8) 9.4 (X3-15.2) 8.0 (2.2-14.6) 16.4 (2.X-15.2) 44.7 (2.2-14.6) 16.3 (X9-92.8)
I
ML 1
Y. K.
I9
m
250.0
2
ML with CRS-M”
A. T.
7
f
101.9
3
ML with CRS-M
K. T.
I2
m
130.3
4
ML II
T. N.
6
m
184.6
5
ML II
N. N.
f
130.3
6
ML I1
w. 0.
f
384.0
Control (n = 18)
MUCOLIPIDOSIS
Name
Age
Case
WITH
II 1
35.5 (20.6-67.3)
Nate. Units, mg/g creatinine. “ The urinary excretion levels of uranic acid in the patients were evaluated by comparing with the age-matched normal values reported by Spranger (22) which are listed in parentheses. ’ CRS-M. macular cherry-red spots and myoclonus syndrome.
URINARY
SIALOGLYCOCONJUGATES
235
the normal upper limits reported by Spranger (22) in most of the patients tested (patients A. T., K. T., T. N., and W. O.), while the patients with ML I (patient Y. K.) and ML II (patient N. N.) excreted slightly increased amounts of uranic acid when compared with the age-matched normal values of Spranger (22). lsofation of Urinary SGC
About 5000 ml of centrifuged urine was applied to an activated granular charcoal column (5 x 60 cm). After washing the column with 5 vol of distilled water, sialic-acid-containing materials were eluted with a mixture of ethanol, pyridine, and distilled water (2: 1:2, by vol). The eluate was concentrated to syrup, and the residue was extracted with three 50-ml portions of distilled water. The extract from normal urine and those from the patients with ML were applied, respectively, to a Dowex 50 (hydrogen form, 200-400 mesh) column (2.5 x 30 cm) jointed to a Dowex I x 2 (acetate form, 200-400 mesh) column (2.5 x 40 cm). After washing the columns with distilled water, sialic-acid-containing materials were eluted from the Dowex 1 x 2 column with 1.0 M acetic acid adjusted to pH 5.3 with pyridine (pyridinium acetate). The eluate was concentrated to dryness, and the residue was extracted with three 20-ml portions of distilled water. (When the volume of the urine specimen was 500 ml or less, the size of the columns was proportionally scaled down.) Gel Filtration
of Urinary SGC on u Sephadex G-25 Column
A 5-ml aliquot of the extract isolated in the previous procedure was applied to a Sephadex G-25 fine column (2.5 x 90 cm) and eluted with 0.1 M pyridinium acetate (pH 5.3). Fractions of 10 ml were collected and scanned for their sialic acid concentration. The urinary SGC were fractionated into 2 major and 1 minor fractions as shown in Fig. 1A. Each fraction was pooled and concentrated, and the residues were dissolved in a small volume of aqueous methanol and precipitated with ethanol and ether. It was suggested by the comparison of the amounts of these fractions that the increased excretion of urinary sialic acid in the patients with ML depended on the excretion levels of 2 fractions designated SGC1 and SGC-1’ (Table 2). Gel Filtration
of SGC-I on a Sephadex G-50 Column
An aqueous solution (5 ml) containing 300 mg of SGC-1 was applied to a Sephadex G-50 fine column (2.5 x 90 cm) and eluted with 0.1 M pyridinium acetate (pH 5.3). Fractions of 10 ml were collected and scanned for their sialic acid and protein concentration. In Fig. IB a representative elution pattern of SGC-1 from the Sephadex G-50 column is shown. Sialic-acid-containing materials were divided into 4 fractions
bLL.2 . L KOSEKI
236 (A) 2.4 2.0
(B)
,)A:, 18::
Tube
number
ET AL.
lC$: 10,
( C]
O.lM
0.25M0.5M
1.0~
*
cu a@ 1.6 P 1.2 ,o
i
/
l
h.
2 0.8
.
0.4
l ii *.
1J
0 20
30
40
(10ml
tube)
FIG. I. Fractionation of urinary sialoglycoconjugates isolated by charcoal column chromatography. (A) A representative gel-filtration pattern of urinary sialoglycoconjugates on Sephadex G-25. Sialic acid concentration of the fractions was determined by the thiobarbituric acid method after mild acid hydrolysis of the eluates with 0.1 N sulfuric acid at 80°C for 60 min. (B) A representative elution pattern of the SGC-1 fraction on Sephadex G-50. Sialic acid and protein concentration of the fractions was determined by the orcinol method (0) and by the method of Lowry et al. (0). respectively. (C) A representative elution pattern of the SGC-ID fraction in Dowex 1 x 2 ion-exchange chromatography. Sialoglycoconjugates were eluted with pyridinium acetate (pH 5.3) with the concentration indicated in the figure. Sialic acid concentration of the fractions was determined by the orcinol method.
TABLE DISTRIBUTION
OF URINARY
2
SIALOGLYCOCONJUGATES
IN SEPHADEX
GEL
FILTRATION
Case number and name Control
1 Y. K.
2 A. T.
3 K. T.
4 T. N.
5 N. N.
6 w. 0.
SGC-1’ SGC-2
64.6 nd 57.7
503.0 56.4 55.8
358.3 156.5 148.7
624.4 230.8 173.1
359.1 82.4 68.9
251.3 60.0 75.8
439. I Ill.0 139.8
SGC-IA SW-IB SW-IC SGC-ID
10.9 20.1 18.1 25.6
44.4 35.4 98.4 290.5
37.3 35.4 44.0 194.2
23.1 57.7 42.3 389.2
41.1 60.2 73.8 150.9
20.8 34.5 40.6 124.7 - ___
32.0 56.2 121.1 180.4 ._ _ .~
Fractions SGC-I
NOW. Units, mg/liter of urine. ” nd. not detectable.
URINARY
237
SIALOGLYCOCONJUGATES
according to their molecular size. The recoveries of each fraction are listed in Table 2. It was indicated by the comparison of the amounts of these fractions that the fraction with the smallest molecular size (SGCID) contributed most to the increase of SGC-I in the patients with ML. Dowex I Ion-Exchange Chromatography of SGC-ID
An aqueous solution containing 300 mg of SGC-ID was applied to a Dowex 1 x 2 (acetate form, 200-400 mesh) column (1.5 x 30 cm), and the column was washed with 200 ml of distilled water. Then sialic-acidcontaining materials were eluted with 4 increasing concentrations of pyridinium acetate (pH 5.3). Fractions of 10 ml were collected and scanned for their sialic acid concentration. Figure IC shows a representative elution pattern of SGC-ID from the Dowex 1 x 2 column. SGC-1D was divided into 8 fractions as indicated in the figure, and was recovered by precipitation with ethanol and ether. As shown in Table 3, the increase of SGC-ID in the patients with ML depended exclusively on the amounts of the fractions eluted with 0.1 M pyridinium acetate (pH 5.3). Chemical Compositions of the Major Fractions of Urinary SGC
The carbohydrate and amino acid compositions of the major fractions of SGC-1D (fractions 0.1 M-A, 0.1 M-B, 0.1 M-C, and 0.25 M-A) were determined by calorimetric and gas-liquid chromatographic analyses. It was indicated by the results (Table 4) that fractions 0.1 M-A and 0.1 MC, which contributed most to the increased urinary excretion of sialic acid. possessed a distinct carbohydrate composition: these were rich in TABLE DISTRIBUTION
3
OF THE SUBFRACTIONS OF SGC-ID IN D~WEX CHROMATOGRAPHY -__~
I x 2 ION-EXCHANGE
Case number and name Subfraction 0.1 M-A 0.1 M-B 0.1 M-c 0.25 M-A 0.25 M-B 0.5 M-A 0.5 M-B 1.0 M
Control
1 Y. K.
2 A. T.
3 K. T.
4 T. N.
5 N. N.
6 w. 0.
11.9 16.1 11.2 17.2 13.2 12.4 7.0 11.1
15.3 0 57.3 15.5 1.4 6.0 2.6 1.9
6.9 1.6 61.0 19.6 3.0 tr 0 0
8.9 9.0 62.6 18.1 tr” tr 0 0
16.5 19.3 32.7 20.6 4.1 4.8 tr 2.0
15.3 14.5 33.4 23.0 tr 7.8 3.9 2.5
17.6 14.4 36.7 11.6 4.6 11.6 0 6.5
Note. Units, (sialic acid/total sialic acid) x 100. ” tr, trace amount.
Y. K. T. N. N. N. w. 0.
Control I 4 5 6
Control 1 4 5 6
0.1 M-c
0.25 M-A
N0fe. Units. mm01/100 g. The chemical compositions ’ tr. trace amount.
T. N. N. N. w. 0.
4 5 6
of sialoglycoconjugates
Y. K. T. N. N. N. w. 0.
Y. K.
Control 1
0.1 M-B
Name
Y. K. T. N. N. N. w. 0.
number
Control 1 4 5 6
Case”
0.1 M-A
Fraction number
CHEMICAL
TABLE
114.4 137.9 124.8 122.5 Ill.8
108.3 88.5 110.4 102.9 108.7
135.2 105.9 110.2 108.4 97.0
112.4 91.9 103.3 105.9 91.7
not been
97.0 115.9 140.1 128.0 83.3
20.9 4.1 1.1 14.1 8.7
have
140.2 141.3 142.9 157.8 140.1
183.2 156.8 165.7 155.5
180.1 168.7 174.5 170.3 164.3
GlcNAc
OF SGC-ID
18.1 0 tf tr 6.8
20.9 16.6 23.3 14.3
55.4 6.5 27.6 25.7 21.6
Fuc
SUBFRACTIONS
the case 2 and 3 patients
63.5 82.9 92.6 80.2 68.3
101.5 135.1 139.5 138.3 114.8
113.3 114.8 112.1 116.3
Man
100.8 110.4 105.7 98.6
4
OF THE MAJOR
Gal
from
COMPOSITIONS
80.9 90.3 90.3 91.6 82.2
14.5 0 0 0 0
determined.
32.1 20.4 0 12.6 32.2
84.4 108.4 89.6 90.8 91.6
78.3 74.4 80.9 69.9
10.7 0 0 0
47.9 72.2 51.3 49.8 48.2
NeuAc
21.6 0 0 0 0
GalNAc ~~~~~
Total
117.1 62.3 56.1 65.3 101.7
47.7 3.7 12.2 10.3 14.9
30.8 39.2 41.4 37.9
48.3 13.0 34.1 30.6 25.9
amino acid
G > r-
x 5 cri E
URINARY
SIALOGLYCOCONJUGATES
239
mannose and glucosamine, and poor in galactosamine and amino acid contents. The major fractions of urinary SGC (SGC-1’ and fraction 0.1 M-C) were analyzed for their oligosaccharide compositions by thin-layer chromatography on a silica gel plate (Kieselgel FZS4, E. Merck, Darnstadt, West Germany), using a mixture of n-propanol, 13 N ammonia, and distilled water (6:2: 1, by vol) as solvent. The chromatograms are shown in Fig. 2. It was shown in the thin-layer chromatograms that monosialyl-orosoN-pentaose (MS-penta) (23) and disialyl-oroso-N-octaose (DS-octa) (24) were common constituents of SGC-1’ and fraction 0.1 M-C in all the patients tested, respectively. A remarkable variation was observed, however, in the color intensity of the reducing spots of monosialyl-oroso-Npentaose II (MS-penta II) (25). The SGC-1’ fractions from the patients with ML II contained approximately equal amounts of MS-penta and MS-penta II. In contrast, the amount of MS-penta II in the SGC-I’ fraction from the patient with ML I was far smaller than that of MSpenta, while the SGC-1’ fractions from the patients with macular cherryred spots and myoclonus syndrome contained trace or negligible amounts of MS-penta II (Fig. 2). DISCUSSION
In the results obtained in the present study it was revealed that there was excessive urinary excretion of SGC in six patients with ML. The urinary SGC from these patients were fractionated into 3 parts according to their molecular size (SGC-1, SGC-I’, and SGC-2). The increased urinary excretion of SGC appears to depend upon the excretion levels of the former 2 fractions. The major component of SGC-1 (fraction 0.1 M-C) is clearly distinguished from others in that it is rich in mannose and glucosamine, and poor in galactosamine and amino acid contents. A similar observation has been reported for the major component of SGC-1’ (23). It was suggested by these observations that the increased urinary excretion of SGC is the result of incomplete catabolism of glycoproteins with an N-glycosidic linkage. In the previous study, we isolated and characterized 3 sialyl-oligosaccharides from the urine of a patient with ML I (patient Y. K.) (23-25). As shown in Fig. 3 these oligosaccharides possess the core structure of the carbohydrate side chains of glycoproteins with an N-glycosidic linkage (26). The sialyl residues of the former two oligosaccharides are linked to galactosyl residues by an a2,6-linkage and that of the latter by an a2,3-linkage. The ol2,6-linked hexasaccharide (MS-penta) is a common constituent of the fraction SGC-1’ in all the patients tested. Likewise, the cY2,6-linked
FIG. 2. Thin-layer chromatograms of the major sialoglycoconjugates isolated from the urine of patients with mucolipidosis. (A. B) Chromatograms of the SGC-I’ fraction. (C D) Chromatograms of the major fraction of 59X-I (fraction 0.1 M-C). MS-penta. monosialyloroso-N-pentaose; DS-octa, disialyl-oroso-N-octaose: MS-penta 11, monosialyl-oroso-Npentaose II: St., standard oligosaccharides; Y. K.. patient with ML I: A. T. and K. T., patients with macular cherry-red spots and myoclonus syndrome; T. N. and N. N.. patients with ML II.
URINARY
241
SIALOGLYCOCONJUGATES
Disialyl-omso-N-rxtse Nemca2,6Gal~1,4GlcNAcBlrZManal,~al,3 NeuAcu2,6GalB1,4Gl~GlcNAcB1,2Manal,6
FIG. 3. The structure of 3 sialyl-oligosaccharides with mucoiipidosis 1.
\
b!an61,4GlcNAc
/
isolated from the urine of a patient
decassacharide (DS-octa) is commonly found in the major fraction of SGC-1 (fraction 0.1 M-C). In the patients with ML II, the color intensity of the reducing spots of MS-penta II is comparable to that of MS-penta. In contrast, the content of MS-penta II in the SGC-1’ fraction from the patient with ML I is far smaller than that of MS-penta, while the SGC1’ fractions from the patients with macular cherry-red spots and myoclonus syndrome contain only a trace amount of MS-penta II. A similar observation has been reported by Strecker and Michalski (27), and they have classified these diseases into 2 groups, sialidosis A and sialidosis B. According to their classification, ML II and ML III are classified into sialidosis A, and ML I and its related disorders into sialidosis B. It has been demonstrated in the recent investigation, however, that the primary lesion of ML II and ML III is not the deficiency of sialidase but the defect in recognition markers required for the intracellular uptake of lysosomal enzymes (28). The increased urinary excretion of sialyl-oligosaccharides observed in the patients with ML II is considered to be caused by this biochemical abnormality. On the other hand, it has been proposed that the primary lesion of ML I and its related disorders is the deficiency of sialidases, therefore, the term “sialidosis” has been proposed for these diseases (1,9). Although we have not determined the sialidase activity, the deficiency of sialidases is probably the primary cause of the increased urinary excretion of sialyl-oligosaccharides observed in the patients with sialidosis (patients Y. K., A. T. and K. T.). Among the present series of experiments, the most remarkable is the fact that there was a marked difference in the excretion levels of the cc2,3-linked hexasaccharide (MS-penta II) between the patient with ML I and those with macular cherry-red spots and myoclonus syndrome. At present the biochemical basis of this dif-
242
KOSEKI
El‘ AI..
ference is unclear, but by this fact we have assumed that the urinary excretion of u2,34inked oligosaccharide is influenced by the difference in the mode of genetic mutation responsible for the biosynthesis of sialidases. SUMMARY Excessive urinary excretion of sialoglycoconjugates was observed in 6 patients with mucolipidosis. Urinary sialoglycoconjugates were divided into 9 fractions by sequential gel filtration on Sephadex G-25 and G-50, and by Dowex I ionexchange chromatography. In a comparative study on these fractions it was shown that the fractions rich in sialyl-oligosaccharide contents contributed to the increased urinary excretion of sialoglycoconjugates. The sialyl-oligosaccharides contained in these fractions possessed the core structure of the carbohydrate side chains of glycoproteins with an Nglycosidic linkage, and the sialyl residues in these oligosaccharides were linked to galactosyl residues by an (w2,3- or a2,6-linkage. In contrast to the latter type oligosaccharides, the excretion levels of the former varied from patient to patient. The patients with ML II excreted the former oligosaccharide comparable to the excretion levels of the latter. In the patient with ML I the excretion levels of cw2,34inked oligosaccharide was far lower than those of a2,6-linked one, and the patients with macular cherry-red spots and myoclonus syndrome excreted only a trace amount of a2,3-linked oligosaccharide. Mechanisms of these different excretion patterns are discussed. ACKNOWLEDGMENTS We are grateful to Professor T. Kitagawa. Dr. R. Minami. and Dr. M. Owada for supplying the urine specimens. We also thank Mr. M. Sasaki for his skillful technical assistance in automatic amino-acid and amino-sugar analysis. This work was supported in part by a research grant from the Ministry of Education, Science and Culture. Japan.
REFERENCES 1. Spranger, J., Gehler, J., and Cantz. M.. Amer. J. Med. Genef. 1, 21 (1977). 2. Orii. T., Minami, R.. Sukegawa, K., Sato, S., Tsugawa, S.. Horino. K., Miura. R.. and Nakao, T., To/z&l J. Exp. Med. 107, 303 (1972). 3. Rapin, I., Goldfischer, S., Katzman, R., Engel, J., and O’Brien, J. S., Ann. Neural. 3, 234 (1977). 4. Kelly, T. E., and Graetz, G., Amer. .I. Med. Gene?. 1, 31 (1977). 5. Thomas, H. H., Tipton. R. E., Ch’ien. L. T., Reynolds, L. W., and Miller, C. S., Clin. Genet. 13, 369 (1978). 6. Wenger, D. A., Tarby. T. J.. and Wharton, C., B&hem. Biophys. Res. Commun. 82, 589 (1978). 7. Maroteaux. P., Humbel. R.. Strecker. G.. Michalski. J.-C.. and Mande, R., Arch. Fr. Pediutr. 35. 819 (1978).
URINARY
SIALOGLYCOCONJUGATES
243
8. Miyatake, T., Yamada, T., Suzuki, M., Pallman, B., Sandhoff, K., Ariga, T., and Atsumi, T., FEBS Lert. 97, 257 (1978). 9. Lowden, J. A., and O’Brien, J. S., Amer. J. Hum. Genet. 31, 1 (1979). 10. Owada, M., Nishitani, O., and Kitagawa, T., in “Proceedings of the 82nd Meeting of Societas Paediatrica Japonica, Tokyo,” p. 229 (1979) (in Japanese). Il. Sakiyama, T., Acta Paediarr. Jupon. 19, 19, 36 (1977) (in Japanese with English summary). 12. Takeuchi, M., Kawamura, Y., Abo, W., Nakamura, F., Minami, R.. Karube, K.. Tsugawa, S., and Nakao. T., J. C/in. Paediarr. Sapporo 27,305 (1979) (in Japanese). 13. BBhm, P., Dauber, St., and Baumeister, L., K&n. Wuchenschr. 32, 289 (1954). 14. Warren. L., J. Biol. Chem. 234, 1971 (1959). 15. Scott, J. A., and Melvin, E. H., Anal. Chem. 25, 1656 (1953). 16. Tamura, Z., Imanari, T., and Arakawa, Y., Proc. Symp. Chem. Physiol. Parhol. 8, 72 (1968) (in Japanese with English summary). 17. Blix, G., Acta Chem. Stand. 2, 467 (1948). 18. Lowry, 0. H., Rosebrough, N. J., Farr. A. L.. and Randa, R. J., J. Biol. Chem. 193, 265 (1951). 19. Bitter, T., and Muir, H. E.. Anal. Biochem. 4, 330 (1962). 20. Koseki, M., Wu, J.-Y., and Tsurumi. K., Fukushima J. Med. Sri. 22, 187 (1977). 21. Ohman, R., Acta Chem. Stand. 21, 1670 (1967). 22. Spranger, J. W., Z. Kinderheilk. IO& 17 (1970). 23. Koseki, M., and Tsurumi. K., Tohoku J. Exp. Med. 124, 361 (1978). 24. Koseki, M., and Tsurumi, K., Tohoku J. Exp. Med. lu1, 39 (1979). 25. Koseki, M., and Tsurumi, K., in “Glycoconjugates. Proceedings of the Fifth International Symposium Kiel. Federal Republic of Germany. September 1979” (R. Schauer, P. Buddecke, M. F. Kramer, J. F. G. Vliegenthalt. and H. Wiegandt. Eds.), pp. 390-391. Thieme, Stuttgart, 1979. 26. Kornfeld, R., and Kornfeld, S., Annu. Rev. Biochem. 45, 217 (1976). 27. Strecker, G., and Michalski, J. C., FEBS Lert. 85, 20 (1978). 28. Neufeld, E. F., Sando. G. N.. Garvin, A. J., and Rome, J. H.. J. Supramol. Struct. 6, 95 (1977).
MEDICINF
27, 2X-243
f lY%)
Urinary Excretion Patterns of Sialoglycoconjugates Mucolipidosis
in Patients with
MASAI KOSEKI,’ MICHIKO EJIRI, KAZUO EJIRI, SHIGERUINO, AND KOICHI TSURUMI* Fukushima Institute of Health and *Department oj.Biochemistry. College. Fukushimu 960. Japan
Fukushima Medical
Received September 8, 1981
In 1977 Spranger et al. (1) observed diminished activity of sialidase (EC 3.2.1.18) and excessive tissue accumulation as well as urinary excretion of sialoglycoconjugates (SGC) in one patient with mucolipidosis (ML) I. Accordingly they first proposed the term “sialidosis” for this disease. Later investigations, however, have demonstrated the presence of several variant forms of sialidase deficiency (2-8). The affected patients do not always show the clinical signs characteristic of ML I. The common clinical signs and biochemical findings are macular cherry-red spots, myoclonus, loss of visual acuity, and the disturbed metabolism of SGC. Recently Lowden and O’Brien (9) classified these diseases into two types on the basis of clinical findings, (type 1) normosomatic group and (type 2) dysmorphic group, the latter group being divided into two subgroups, juvenile onset and infantile onset. Although the deficiency of sialidase is the basic defect of these diseases, the biochemical basis for the different clinical types of sialidosis remains unclear. The results of a comparative study of the excretion patterns of sialyl-oligosaccharides in urine of patients with several types of ML are dealt with in this report. MATERIALS AND METHODS Urine specimens. Pooled urine (5000 ml) from a patient with ML I (Y. K., a lPyear-old boy) was supplied by Dr. R. Minami, Sapporo Medical College, Sapporo. The clinical and cytological studies on this ’ To whom correspondence should be addressed: Fukushima Institute of Health, 16-6 Mitouchi, Hokida, Fukushima 960. Japan. 232 0006-2944/82/020232-12$02.00/O Copyright All rights
8 1982 by Academic Press. Inc. of reproductmn in any form recerwd
URINARY SIALOGLYCOCONJUCATES
233
patient have been reported by Orii et al. (2). Urine specimens from 2 subjects with hypersialosiduria, myoclonus, and macular cherry-red spots (a 7-year-old girl, A. T., 78 ml, and her 12-year-old brother, K. T., 115 ml), were supplied by Professor T. Kitagawa and Dr. M. Owada, Nihon University School of Medicine, Tokyo. The clinical study on these patients has been reported by Owada et al. (IO). Urine specimens from 2 subjects with ML II (I-cell disease), a 6-year-old boy (T. N., 450 ml) and his 1l-year-old sister (N. N., 530 ml), were supplied by Professor T. Kitagawa and Dr. M. Owada. The clinical and biochemical studies on these patients have been reported by Sakiyama (11). A urine specimen (450 ml) from a patient with ML II (W. O., a l-year-old girl) was supplied by Dr. R. Minami. The clinical study on this patient has been reported by Takeuchi et al. (12). Normal human urine (16,000 ml) was collected from young healthy boys ranging from 10 to 11 years of age and was used for the isolation and fractionation of urinary SGC. Morning urine was collected from 10 healthy men and 8 healthy women and was used for the determination of normal excretion levels of bound sialic acid. All urine specimens were stored frozen until used. Analytical methods. Total sialic acid was determined by the orcinol method of Bohm et al. (13), and free sialic acid by the thiobarbituric acid method of Warren (14). The results were expressed as N-acetylneuraminic acid (NeuAc). Total neutral sugars were determined by the anthrone method (15) using a mixture of equal amounts of galactose and mannose as standard. The molar compositions of neutral sugars were determined by gas-liquid chromatography as their alditol trifluoroacetates. The samples for the assay were hydrolyzed with 2.5 N trifluoroacetic acid at 100°C for 5 hr. The dried hydrolyzates were reduced with sodium borohydride, trifluoroacetylated by the method of Tamura et al. (16) and analyzed by gas-liquid chromatography on a glass column (3 mm x 200 cm) packed with 3% XF-1105 on Gaschrom Q (SO-100 mesh). The column temperature was programmed from 115 to 145°C at a rate of l”C/min. Total hexosamines were determined by the Elson-Morgan reaction described by Blix (17), and the samples for the assay were hydrolyzed with 2 N hydrochloric acid at 100°C for 14 hr. Total protein was determined by the method of Lowry et al. (18) using human serum protein as standard. Uranic acid was determined by the carbazole method of Bitter and Muir (19) using glucuronic acid as standard. The compositions of amino acids and amino sugars were determined on a JLC-5AH automatic amino acid analyzer. The samples for the assay were hydrolyzed with 6 N hydrochloric acid at 110°C for 22 hr. Urinary SGC were isolated and determined by the method described by Koseki et al. (20). For the determination of glycosaminoglycans, the urine samples were diluted to obtain a specific gravity of 1.020 or less and adjusted to pH
34
KOSEKI
ET AL.
6.0. To 10 ml of these samples was added 0.5 ml of 5% cetylpyridinium chloride. After standing overnight at 4°C. the precipitates formed were separated, washed with NaGsaturated ethanol. and dissolved in 3 ml of 0.01 N sodium hydroxide. Uranic acid contents of these samples were determined by the carbazole method. Thin-layer chromatography of sialyl-oligosaccharides was performed according to the method of ohman (21), and spots of reducing sugars were visualized by spraying the aniline hydrogen phthalate reagent. EXPERIMENTS
AND RESULTS
Determination qf Urirtary Sialic Acid and Uranic Acid Urinary sialic acid in patients with ML and in healthy individuals was determined by the charcoal adsorption method described in our previous paper (20). Urinary glycosaminoglycans were precipitated with cetylpyridinium chloride, and uranic acid contents of the precipitates were determined by the carbazole method. The results are listed in Table I. As shown in the table, increased urinary excretion of sialic acid was observed in the patients with so-called sialidosis. The patients with Icell disease also excreted increased amounts of sialic acid, the excretion levels of which were indistinguishable from those of the patients with sialidosis. In contrast, the excretion levels of uranic acid were within TABLE URINARY
EXCRETION
OF SIALIC ACID
AND
I
URONK
ACID
IN PATIENTS
Syndrome
(years)
Sex
Sialic acid
Uranic acid“ 13.3 (IA- 2.8) 9.4 (X3-15.2) 8.0 (2.2-14.6) 16.4 (2.X-15.2) 44.7 (2.2-14.6) 16.3 (X9-92.8)
I
ML 1
Y. K.
I9
m
250.0
2
ML with CRS-M”
A. T.
7
f
101.9
3
ML with CRS-M
K. T.
I2
m
130.3
4
ML II
T. N.
6
m
184.6
5
ML II
N. N.
f
130.3
6
ML I1
w. 0.
f
384.0
Control (n = 18)
MUCOLIPIDOSIS
Name
Age
Case
WITH
II 1
35.5 (20.6-67.3)
Nate. Units, mg/g creatinine. “ The urinary excretion levels of uranic acid in the patients were evaluated by comparing with the age-matched normal values reported by Spranger (22) which are listed in parentheses. ’ CRS-M. macular cherry-red spots and myoclonus syndrome.
URINARY
SIALOGLYCOCONJUGATES
235
the normal upper limits reported by Spranger (22) in most of the patients tested (patients A. T., K. T., T. N., and W. O.), while the patients with ML I (patient Y. K.) and ML II (patient N. N.) excreted slightly increased amounts of uranic acid when compared with the age-matched normal values of Spranger (22). lsofation of Urinary SGC
About 5000 ml of centrifuged urine was applied to an activated granular charcoal column (5 x 60 cm). After washing the column with 5 vol of distilled water, sialic-acid-containing materials were eluted with a mixture of ethanol, pyridine, and distilled water (2: 1:2, by vol). The eluate was concentrated to syrup, and the residue was extracted with three 50-ml portions of distilled water. The extract from normal urine and those from the patients with ML were applied, respectively, to a Dowex 50 (hydrogen form, 200-400 mesh) column (2.5 x 30 cm) jointed to a Dowex I x 2 (acetate form, 200-400 mesh) column (2.5 x 40 cm). After washing the columns with distilled water, sialic-acid-containing materials were eluted from the Dowex 1 x 2 column with 1.0 M acetic acid adjusted to pH 5.3 with pyridine (pyridinium acetate). The eluate was concentrated to dryness, and the residue was extracted with three 20-ml portions of distilled water. (When the volume of the urine specimen was 500 ml or less, the size of the columns was proportionally scaled down.) Gel Filtration
of Urinary SGC on u Sephadex G-25 Column
A 5-ml aliquot of the extract isolated in the previous procedure was applied to a Sephadex G-25 fine column (2.5 x 90 cm) and eluted with 0.1 M pyridinium acetate (pH 5.3). Fractions of 10 ml were collected and scanned for their sialic acid concentration. The urinary SGC were fractionated into 2 major and 1 minor fractions as shown in Fig. 1A. Each fraction was pooled and concentrated, and the residues were dissolved in a small volume of aqueous methanol and precipitated with ethanol and ether. It was suggested by the comparison of the amounts of these fractions that the increased excretion of urinary sialic acid in the patients with ML depended on the excretion levels of 2 fractions designated SGC1 and SGC-1’ (Table 2). Gel Filtration
of SGC-I on a Sephadex G-50 Column
An aqueous solution (5 ml) containing 300 mg of SGC-1 was applied to a Sephadex G-50 fine column (2.5 x 90 cm) and eluted with 0.1 M pyridinium acetate (pH 5.3). Fractions of 10 ml were collected and scanned for their sialic acid and protein concentration. In Fig. IB a representative elution pattern of SGC-1 from the Sephadex G-50 column is shown. Sialic-acid-containing materials were divided into 4 fractions
bLL.2 . L KOSEKI
236 (A) 2.4 2.0
(B)
,)A:, 18::
Tube
number
ET AL.
lC$: 10,
( C]
O.lM
0.25M0.5M
1.0~
*
cu a@ 1.6 P 1.2 ,o
i
/
l
h.
2 0.8
.
0.4
l ii *.
1J
0 20
30
40
(10ml
tube)
FIG. I. Fractionation of urinary sialoglycoconjugates isolated by charcoal column chromatography. (A) A representative gel-filtration pattern of urinary sialoglycoconjugates on Sephadex G-25. Sialic acid concentration of the fractions was determined by the thiobarbituric acid method after mild acid hydrolysis of the eluates with 0.1 N sulfuric acid at 80°C for 60 min. (B) A representative elution pattern of the SGC-1 fraction on Sephadex G-50. Sialic acid and protein concentration of the fractions was determined by the orcinol method (0) and by the method of Lowry et al. (0). respectively. (C) A representative elution pattern of the SGC-ID fraction in Dowex 1 x 2 ion-exchange chromatography. Sialoglycoconjugates were eluted with pyridinium acetate (pH 5.3) with the concentration indicated in the figure. Sialic acid concentration of the fractions was determined by the orcinol method.
TABLE DISTRIBUTION
OF URINARY
2
SIALOGLYCOCONJUGATES
IN SEPHADEX
GEL
FILTRATION
Case number and name Control
1 Y. K.
2 A. T.
3 K. T.
4 T. N.
5 N. N.
6 w. 0.
SGC-1’ SGC-2
64.6 nd 57.7
503.0 56.4 55.8
358.3 156.5 148.7
624.4 230.8 173.1
359.1 82.4 68.9
251.3 60.0 75.8
439. I Ill.0 139.8
SGC-IA SW-IB SW-IC SGC-ID
10.9 20.1 18.1 25.6
44.4 35.4 98.4 290.5
37.3 35.4 44.0 194.2
23.1 57.7 42.3 389.2
41.1 60.2 73.8 150.9
20.8 34.5 40.6 124.7 - ___
32.0 56.2 121.1 180.4 ._ _ .~
Fractions SGC-I
NOW. Units, mg/liter of urine. ” nd. not detectable.
URINARY
237
SIALOGLYCOCONJUGATES
according to their molecular size. The recoveries of each fraction are listed in Table 2. It was indicated by the comparison of the amounts of these fractions that the fraction with the smallest molecular size (SGCID) contributed most to the increase of SGC-I in the patients with ML. Dowex I Ion-Exchange Chromatography of SGC-ID
An aqueous solution containing 300 mg of SGC-ID was applied to a Dowex 1 x 2 (acetate form, 200-400 mesh) column (1.5 x 30 cm), and the column was washed with 200 ml of distilled water. Then sialic-acidcontaining materials were eluted with 4 increasing concentrations of pyridinium acetate (pH 5.3). Fractions of 10 ml were collected and scanned for their sialic acid concentration. Figure IC shows a representative elution pattern of SGC-ID from the Dowex 1 x 2 column. SGC-1D was divided into 8 fractions as indicated in the figure, and was recovered by precipitation with ethanol and ether. As shown in Table 3, the increase of SGC-ID in the patients with ML depended exclusively on the amounts of the fractions eluted with 0.1 M pyridinium acetate (pH 5.3). Chemical Compositions of the Major Fractions of Urinary SGC
The carbohydrate and amino acid compositions of the major fractions of SGC-1D (fractions 0.1 M-A, 0.1 M-B, 0.1 M-C, and 0.25 M-A) were determined by calorimetric and gas-liquid chromatographic analyses. It was indicated by the results (Table 4) that fractions 0.1 M-A and 0.1 MC, which contributed most to the increased urinary excretion of sialic acid. possessed a distinct carbohydrate composition: these were rich in TABLE DISTRIBUTION
3
OF THE SUBFRACTIONS OF SGC-ID IN D~WEX CHROMATOGRAPHY -__~
I x 2 ION-EXCHANGE
Case number and name Subfraction 0.1 M-A 0.1 M-B 0.1 M-c 0.25 M-A 0.25 M-B 0.5 M-A 0.5 M-B 1.0 M
Control
1 Y. K.
2 A. T.
3 K. T.
4 T. N.
5 N. N.
6 w. 0.
11.9 16.1 11.2 17.2 13.2 12.4 7.0 11.1
15.3 0 57.3 15.5 1.4 6.0 2.6 1.9
6.9 1.6 61.0 19.6 3.0 tr 0 0
8.9 9.0 62.6 18.1 tr” tr 0 0
16.5 19.3 32.7 20.6 4.1 4.8 tr 2.0
15.3 14.5 33.4 23.0 tr 7.8 3.9 2.5
17.6 14.4 36.7 11.6 4.6 11.6 0 6.5
Note. Units, (sialic acid/total sialic acid) x 100. ” tr, trace amount.
Y. K. T. N. N. N. w. 0.
Control I 4 5 6
Control 1 4 5 6
0.1 M-c
0.25 M-A
N0fe. Units. mm01/100 g. The chemical compositions ’ tr. trace amount.
T. N. N. N. w. 0.
4 5 6
of sialoglycoconjugates
Y. K. T. N. N. N. w. 0.
Y. K.
Control 1
0.1 M-B
Name
Y. K. T. N. N. N. w. 0.
number
Control 1 4 5 6
Case”
0.1 M-A
Fraction number
CHEMICAL
TABLE
114.4 137.9 124.8 122.5 Ill.8
108.3 88.5 110.4 102.9 108.7
135.2 105.9 110.2 108.4 97.0
112.4 91.9 103.3 105.9 91.7
not been
97.0 115.9 140.1 128.0 83.3
20.9 4.1 1.1 14.1 8.7
have
140.2 141.3 142.9 157.8 140.1
183.2 156.8 165.7 155.5
180.1 168.7 174.5 170.3 164.3
GlcNAc
OF SGC-ID
18.1 0 tf tr 6.8
20.9 16.6 23.3 14.3
55.4 6.5 27.6 25.7 21.6
Fuc
SUBFRACTIONS
the case 2 and 3 patients
63.5 82.9 92.6 80.2 68.3
101.5 135.1 139.5 138.3 114.8
113.3 114.8 112.1 116.3
Man
100.8 110.4 105.7 98.6
4
OF THE MAJOR
Gal
from
COMPOSITIONS
80.9 90.3 90.3 91.6 82.2
14.5 0 0 0 0
determined.
32.1 20.4 0 12.6 32.2
84.4 108.4 89.6 90.8 91.6
78.3 74.4 80.9 69.9
10.7 0 0 0
47.9 72.2 51.3 49.8 48.2
NeuAc
21.6 0 0 0 0
GalNAc ~~~~~
Total
117.1 62.3 56.1 65.3 101.7
47.7 3.7 12.2 10.3 14.9
30.8 39.2 41.4 37.9
48.3 13.0 34.1 30.6 25.9
amino acid
G > r-
x 5 cri E
URINARY
SIALOGLYCOCONJUGATES
239
mannose and glucosamine, and poor in galactosamine and amino acid contents. The major fractions of urinary SGC (SGC-1’ and fraction 0.1 M-C) were analyzed for their oligosaccharide compositions by thin-layer chromatography on a silica gel plate (Kieselgel FZS4, E. Merck, Darnstadt, West Germany), using a mixture of n-propanol, 13 N ammonia, and distilled water (6:2: 1, by vol) as solvent. The chromatograms are shown in Fig. 2. It was shown in the thin-layer chromatograms that monosialyl-orosoN-pentaose (MS-penta) (23) and disialyl-oroso-N-octaose (DS-octa) (24) were common constituents of SGC-1’ and fraction 0.1 M-C in all the patients tested, respectively. A remarkable variation was observed, however, in the color intensity of the reducing spots of monosialyl-oroso-Npentaose II (MS-penta II) (25). The SGC-1’ fractions from the patients with ML II contained approximately equal amounts of MS-penta and MS-penta II. In contrast, the amount of MS-penta II in the SGC-I’ fraction from the patient with ML I was far smaller than that of MSpenta, while the SGC-1’ fractions from the patients with macular cherryred spots and myoclonus syndrome contained trace or negligible amounts of MS-penta II (Fig. 2). DISCUSSION
In the results obtained in the present study it was revealed that there was excessive urinary excretion of SGC in six patients with ML. The urinary SGC from these patients were fractionated into 3 parts according to their molecular size (SGC-1, SGC-I’, and SGC-2). The increased urinary excretion of SGC appears to depend upon the excretion levels of the former 2 fractions. The major component of SGC-1 (fraction 0.1 M-C) is clearly distinguished from others in that it is rich in mannose and glucosamine, and poor in galactosamine and amino acid contents. A similar observation has been reported for the major component of SGC-1’ (23). It was suggested by these observations that the increased urinary excretion of SGC is the result of incomplete catabolism of glycoproteins with an N-glycosidic linkage. In the previous study, we isolated and characterized 3 sialyl-oligosaccharides from the urine of a patient with ML I (patient Y. K.) (23-25). As shown in Fig. 3 these oligosaccharides possess the core structure of the carbohydrate side chains of glycoproteins with an N-glycosidic linkage (26). The sialyl residues of the former two oligosaccharides are linked to galactosyl residues by an a2,6-linkage and that of the latter by an a2,3-linkage. The ol2,6-linked hexasaccharide (MS-penta) is a common constituent of the fraction SGC-1’ in all the patients tested. Likewise, the cY2,6-linked
FIG. 2. Thin-layer chromatograms of the major sialoglycoconjugates isolated from the urine of patients with mucolipidosis. (A. B) Chromatograms of the SGC-I’ fraction. (C D) Chromatograms of the major fraction of 59X-I (fraction 0.1 M-C). MS-penta. monosialyloroso-N-pentaose; DS-octa, disialyl-oroso-N-octaose: MS-penta 11, monosialyl-oroso-Npentaose II: St., standard oligosaccharides; Y. K.. patient with ML I: A. T. and K. T., patients with macular cherry-red spots and myoclonus syndrome; T. N. and N. N.. patients with ML II.
URINARY
241
SIALOGLYCOCONJUGATES
Disialyl-omso-N-rxtse Nemca2,6Gal~1,4GlcNAcBlrZManal,~al,3 NeuAcu2,6GalB1,4Gl~GlcNAcB1,2Manal,6
FIG. 3. The structure of 3 sialyl-oligosaccharides with mucoiipidosis 1.
\
b!an61,4GlcNAc
/
isolated from the urine of a patient
decassacharide (DS-octa) is commonly found in the major fraction of SGC-1 (fraction 0.1 M-C). In the patients with ML II, the color intensity of the reducing spots of MS-penta II is comparable to that of MS-penta. In contrast, the content of MS-penta II in the SGC-1’ fraction from the patient with ML I is far smaller than that of MS-penta, while the SGC1’ fractions from the patients with macular cherry-red spots and myoclonus syndrome contain only a trace amount of MS-penta II. A similar observation has been reported by Strecker and Michalski (27), and they have classified these diseases into 2 groups, sialidosis A and sialidosis B. According to their classification, ML II and ML III are classified into sialidosis A, and ML I and its related disorders into sialidosis B. It has been demonstrated in the recent investigation, however, that the primary lesion of ML II and ML III is not the deficiency of sialidase but the defect in recognition markers required for the intracellular uptake of lysosomal enzymes (28). The increased urinary excretion of sialyl-oligosaccharides observed in the patients with ML II is considered to be caused by this biochemical abnormality. On the other hand, it has been proposed that the primary lesion of ML I and its related disorders is the deficiency of sialidases, therefore, the term “sialidosis” has been proposed for these diseases (1,9). Although we have not determined the sialidase activity, the deficiency of sialidases is probably the primary cause of the increased urinary excretion of sialyl-oligosaccharides observed in the patients with sialidosis (patients Y. K., A. T. and K. T.). Among the present series of experiments, the most remarkable is the fact that there was a marked difference in the excretion levels of the cc2,3-linked hexasaccharide (MS-penta II) between the patient with ML I and those with macular cherry-red spots and myoclonus syndrome. At present the biochemical basis of this dif-
242
KOSEKI
El‘ AI..
ference is unclear, but by this fact we have assumed that the urinary excretion of u2,34inked oligosaccharide is influenced by the difference in the mode of genetic mutation responsible for the biosynthesis of sialidases. SUMMARY Excessive urinary excretion of sialoglycoconjugates was observed in 6 patients with mucolipidosis. Urinary sialoglycoconjugates were divided into 9 fractions by sequential gel filtration on Sephadex G-25 and G-50, and by Dowex I ionexchange chromatography. In a comparative study on these fractions it was shown that the fractions rich in sialyl-oligosaccharide contents contributed to the increased urinary excretion of sialoglycoconjugates. The sialyl-oligosaccharides contained in these fractions possessed the core structure of the carbohydrate side chains of glycoproteins with an Nglycosidic linkage, and the sialyl residues in these oligosaccharides were linked to galactosyl residues by an (w2,3- or a2,6-linkage. In contrast to the latter type oligosaccharides, the excretion levels of the former varied from patient to patient. The patients with ML II excreted the former oligosaccharide comparable to the excretion levels of the latter. In the patient with ML I the excretion levels of cw2,34inked oligosaccharide was far lower than those of a2,6-linked one, and the patients with macular cherry-red spots and myoclonus syndrome excreted only a trace amount of a2,3-linked oligosaccharide. Mechanisms of these different excretion patterns are discussed. ACKNOWLEDGMENTS We are grateful to Professor T. Kitagawa. Dr. R. Minami. and Dr. M. Owada for supplying the urine specimens. We also thank Mr. M. Sasaki for his skillful technical assistance in automatic amino-acid and amino-sugar analysis. This work was supported in part by a research grant from the Ministry of Education, Science and Culture. Japan.
REFERENCES 1. Spranger, J., Gehler, J., and Cantz. M.. Amer. J. Med. Genef. 1, 21 (1977). 2. Orii. T., Minami, R.. Sukegawa, K., Sato, S., Tsugawa, S.. Horino. K., Miura. R.. and Nakao, T., To/z&l J. Exp. Med. 107, 303 (1972). 3. Rapin, I., Goldfischer, S., Katzman, R., Engel, J., and O’Brien, J. S., Ann. Neural. 3, 234 (1977). 4. Kelly, T. E., and Graetz, G., Amer. .I. Med. Gene?. 1, 31 (1977). 5. Thomas, H. H., Tipton. R. E., Ch’ien. L. T., Reynolds, L. W., and Miller, C. S., Clin. Genet. 13, 369 (1978). 6. Wenger, D. A., Tarby. T. J.. and Wharton, C., B&hem. Biophys. Res. Commun. 82, 589 (1978). 7. Maroteaux. P., Humbel. R.. Strecker. G.. Michalski. J.-C.. and Mande, R., Arch. Fr. Pediutr. 35. 819 (1978).
URINARY
SIALOGLYCOCONJUGATES
243
8. Miyatake, T., Yamada, T., Suzuki, M., Pallman, B., Sandhoff, K., Ariga, T., and Atsumi, T., FEBS Lert. 97, 257 (1978). 9. Lowden, J. A., and O’Brien, J. S., Amer. J. Hum. Genet. 31, 1 (1979). 10. Owada, M., Nishitani, O., and Kitagawa, T., in “Proceedings of the 82nd Meeting of Societas Paediatrica Japonica, Tokyo,” p. 229 (1979) (in Japanese). Il. Sakiyama, T., Acta Paediarr. Jupon. 19, 19, 36 (1977) (in Japanese with English summary). 12. Takeuchi, M., Kawamura, Y., Abo, W., Nakamura, F., Minami, R.. Karube, K.. Tsugawa, S., and Nakao. T., J. C/in. Paediarr. Sapporo 27,305 (1979) (in Japanese). 13. BBhm, P., Dauber, St., and Baumeister, L., K&n. Wuchenschr. 32, 289 (1954). 14. Warren. L., J. Biol. Chem. 234, 1971 (1959). 15. Scott, J. A., and Melvin, E. H., Anal. Chem. 25, 1656 (1953). 16. Tamura, Z., Imanari, T., and Arakawa, Y., Proc. Symp. Chem. Physiol. Parhol. 8, 72 (1968) (in Japanese with English summary). 17. Blix, G., Acta Chem. Stand. 2, 467 (1948). 18. Lowry, 0. H., Rosebrough, N. J., Farr. A. L.. and Randa, R. J., J. Biol. Chem. 193, 265 (1951). 19. Bitter, T., and Muir, H. E.. Anal. Biochem. 4, 330 (1962). 20. Koseki, M., Wu, J.-Y., and Tsurumi. K., Fukushima J. Med. Sri. 22, 187 (1977). 21. Ohman, R., Acta Chem. Stand. 21, 1670 (1967). 22. Spranger, J. W., Z. Kinderheilk. IO& 17 (1970). 23. Koseki, M., and Tsurumi. K., Tohoku J. Exp. Med. 124, 361 (1978). 24. Koseki, M., and Tsurumi, K., Tohoku J. Exp. Med. lu1, 39 (1979). 25. Koseki, M., and Tsurumi, K., in “Glycoconjugates. Proceedings of the Fifth International Symposium Kiel. Federal Republic of Germany. September 1979” (R. Schauer, P. Buddecke, M. F. Kramer, J. F. G. Vliegenthalt. and H. Wiegandt. Eds.), pp. 390-391. Thieme, Stuttgart, 1979. 26. Kornfeld, R., and Kornfeld, S., Annu. Rev. Biochem. 45, 217 (1976). 27. Strecker, G., and Michalski, J. C., FEBS Lert. 85, 20 (1978). 28. Neufeld, E. F., Sando. G. N.. Garvin, A. J., and Rome, J. H.. J. Supramol. Struct. 6, 95 (1977).