Ganglioside sialidase distribution in Mucolipidosis type IV cultured fibroblasts

ARCHIVES OF BIOCHEMISTRY Vol. 241, No. 2, September,

AND BIOPHYSICS pp. 602-607, 1985

Ganglioside

Sialidase Distribution in Mucolipidosis Type IV Cultured Fibroblasts

MARCIA Department

of Human

Genetics,

ZEIGLER Hadassah

AND

Medical

Received

GIDEON Center,

April

P.O. Box

BACH’ 12000, 91120 Jerusalem,

Ismel

9, 1985

The subcellular distribution of ganglioside sialidase in Mucolipidosis IV (ML IV) cells was characterized by a series of Percoll gradients. Similar to normal cells, the enzyme cosedimented with plasma membrane markers, although this activity was reduced and exhibited decreased solubility in ML IV cells. Only trace amounts of ganglioside sialidase (<5%) was found in the lysosomes of normal cells. This activity was apparently reduced in ML IV cells but its minute activity in controls excluded further characterization of these differences. Plasma membranes on 6.7 and 5.6% Percoll gradients were biomodally distributed. Ganglioside sialidase in normal cells was found to be in both the heavier and the lighter membrane fractions, whereas the enzyme in ML IV cells was associated mainly with the denser membrane fraction. These data indicate that the enzyme in ML IV cells is characteristically different from normal in that it exhibits reduced activity and solubility and a different plasma membrane distribution. 0 1985 Academic Press, Inc.

Mucolipidosis type IV (ML IV)’ is an autosomal recessive lysosomal storage disorder, first recognized in 1974 (1). Clinically it is characterized by bilateral corneal clouding, slow progressive psychomotor retardation, and no organomegalies (l-4). Patients known to date range in age from 9 months to the mid twenties. As in other mucolipidoses there is storage of lipids, in this instance gangliosides (3, 5, 6) and phospholipids (7), together with water-soluble substances, namely acid mucopolysaccharides (6, 8). Recently we have shown a partial deficiency of soluble gangliosde sialidase in ML IV fibroblasts, as well as in cultured amniotic fluid cells (6, 9), and intermediate activity between patients and controls in obligate heterozygotes. This was later confirmed by others (10, 11). At that time we speculated 1 To whom ‘Abbreviation

correspondence used: ML

that this deficiency was only partial since the absence of a specific ganglioside sialidase may have been masked by the presence of other isozymes not affected in ML IV. In an attempt to localize ganglioside sialidase activity to the various subcellular organelles of cultured skin fibroblasts we fractionated normal cells on a 30% colloidal silica gradient (Percoll), which allows for the clear separation of a dense lysosomal fraction from other cell organelles (12). According to these studies, ganglioside sialidase was located mainly in plasma membranes, and only trace activity of this enzyme was found in the lysosomes (13). The present investigation studies the subcellular distribution of ganglioside sialidase in ML IV fibroblasts as determined by a series of self-generating gradients of different starting densities of Percoll. Similar to controls, ganglioside sialidase in ML IV fibroblasts cosedimented with plasma membranes, but it

should be addressed. IV, mucolipidosis type

IV.

0003-9861/85 Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

602

G.ANGLIOSIDE

exhibited solubility, tribution.

SIALIDASE

IN

consistently reduced activity and an altered membrane

MATIERIALS

AND

MUCOLIPIDOSIS

and dis-

METHODS

Cell culture. ‘Cultured skin fibroblasts from three unrelated, 2- to I-year-old ML IV patients and three normal control,s were propagated in 150-cm2 plastic tissue culture flasks (Falcon) and harvested by trypsinization 14 da.ys after the last transplant. For each gradient, cells from three flasks were collected (7-8 mg protein). Cell maintenance has heen described previously (8, 9). SubceUular fractimutim Subcellular fractionation was performed on a self-generating gradient of Percoll (Pharmacia, Uppsala, Sweden), using 30% Percoll (starting density of 1.065-1.069 g/ml), as described by Rome et al. (12) and Zeigler and Bach (13). By this method lysosomes are distributed in two fractions: one found at a density of 1.085 g/ml and a second peak at a density of 1.11 g/ml. The “light” lysosomes are located in the buoyant band in close proximity to the plasma membrane fragments (density, 1.080 g/ml), golgi apparatus (density, 1.090 g/ml), and the same density as microsomes. In some experiments the dense lysosomes and the buoyant fractions were pooled separately, diluted 1:l (v/v) with physiological saline, and concentrated by centrifugation at 100,OOOg for 1.5 h (4°C). The particles that were layered above the Percoll were collected, pack’ed by centrifugation for 30 min at lOO,OOOg, and resuspended in 0.5 ml HaO. Attempts to solubilize the enzyme from these particles were carried out by ultrasonication for 15 s (Braunson Sonicator 33OS, small probe; B. Braun, West Germany), in the presence of 0.1% Triton X-100 (Sigma Chemicals). Fractionation using 6.7 or 5.6% Percoll was performed according to Sahagian and Neufeld (27) with the following modifications: 6-7 mg protein of postnuclear suspension was layered over 30 ml Percoll with a 4-ml cushion of 2.5 M sucrose in a quick-seal centrifuge tube (Beckman). The gradient was generated by centrifugation in a Beckman ultracentrifuge (Model L8-55) using a Beckman VTi50 vertical rotor at a speed of 20,000 rpm for 75 min with slow acceleration and deceleration. By these gradients lysosomes formed only one peak separated from all other organelles. Plasma membranes and microsomes exhibited a bimodal distribution. Using the 5.6% Percoll, plasma membranes were partially separated from the microsome fraction. These fractionations were performed in all the mentioned cell lines, and each experiment was repeated at least twice; the results are presented as representative data. The recovery of the various enzyme markers in all experiments was 75-80% of the starting activity.

TYPE

IV

FIBROBLASTS

603

Enzyme assays. Ganglioside sialidase was determined by two different techniques: (a) calorimetric determination [Warren (14)] of the released sialic acid from purified GMIl ganglioside or mixed bovine brain gangliosides (Sigma type II) as previously described (13); (b) determination of tritrium-labeled released sialic acid. Radioactively labeled sialic acid (tritiated 5-acetamido-3,5 dideoxy-L-arabinoheptulosonic acid) of purified GMa was prepared by periodate oxidation and reduction with sodium borotritite according to Veh and Schauer (15). Enzyme incubations were carried out with 20-30 pg [3H]GM3 (approximately 100,000 cpm), 0.1-0.3 mg protein, in 0.1 M acetate buffer, pH 4.0, and a final concentration of 0.1% Triton X-100 (Sigma Chemicals) in a volume of 0.2 ml at 37°C for l-4 h. The released tritriumlabeled sialic acid was determined according to Veh and Sander (16). The radioactive labeling of GM3 did not alter the activity of the sialidase against the substrate [see also Ref. (IS)]. Glycoprotein sialidase was determined using neuraminlactose (Sigma type II) as substrate (9). The determination of 5’-nucleotidase (EC 3.1.2.5, plasma membrane marker), a-mannosidase (EC 3.2.1.14), and @-hexosaminidase (EC 3.2.1.30 lysosomal markers) were described previously (17). Na+/K+ ATPase (EC 3.6.1.3, second plasma membrane marker) was determined according to Post and Sen (18). Arylsulfatase C (EC 3.16.1, microsomal marker) was determined using I-methylumbelliferyl sulfate as substrate according to Meyer et al (19). Galactosyl transferase (EC 2.4.1.38, golgi apparatus marker) was determined according to Rome et al (12). RESULTS

The distribution of ganglioside sialidase in normal and ML IV fibroblasts throughout the gradient generated by 30% Percoll is shown in Fig. 1. Most of the ganglisoside sialidase in control and ML IV fibroblasts is associated with the plasma membrane fraction as its distribution runs parallel to 5’-nucleotidase, with a peak activity at a density of 1.080 g/ml. Only trace activity (-4% of total activity) of ganglioside sialidase could be detected in the dense lysosomal fraction of normal fibroblasts, and this activity was almost undetectable in ML IV. However, the minute enzyme activity in the lysosomal fraction made its accurate quantitation extremely difficult and thus precluded any further meaningful characterization. Even pooling and concentration of the dense lysosomal

604

9 O48 (62032 38 40 44 411 50 80 $2

12

24

28

Fraction

52

Number

FIG. 1. Distribution pattern of ganglioside sialidase in normal (A) and ML IV (B) fibroblast postnuclear supernatant on 30% Percoll (starting density). 0, @-Hexosaminidase (lysosomal marker); 0, ganglioside sialidase. The arrows indicate the peak activity of 1. galactosyl transferase, 2. arylsulfatase C, and 3. 5’-nucleotidase. The distribution and the location of the peak activities of the different markers are identical in A and B. a-Mannosidase showed distribution similar to P-hexosaminidase.

fraction two- to threefold did not enable further elaboration either in the control or in ML IV cells. The ganglioside sialidase activity in the pooled and concentrated buoyant fraction of ML IV was reduced as compared to controls (approximately 50% of control values). Following the concentration of the buoyant fraction and partial solubilization of the enzyme from this fraction by ultrasonication in the presence of Triton X-100, significant differences between the two genotypes became apparent (Table I). Under these conditions about 24% of the ganglioside sialidase from normal cells could be solubilized, similar in solubility to the plasma membrane marker, 5’-nucleotidase, while only about 6% ganglioside sialidase could be solubilized from the buoyant fraction of ML IV fibroblasts. Neuraminlactose sialidase in ML IV fibroblasts was found mainly in the dense lysosomal fraction (data not shown), in accordance with our previous results in normal fibroblasts (13). However, this activity in ML IV was consistently elevated at least 1.5-fold over control values. To study in greater detail the distribution of ganglioside sialidase in the plasma membrane, ML IV and normal control

cells were fractionated on a 6.7% Percoll gradient (Fig. 2). On this gradient the plasma membrane fragments and microsomes could be separated from other cellular organelles and exhibited a bimodal distribution. The separation of microsomes into “light” and “heavy” subfractions is not surprising and has been previously reported (20). A similar distribution to

TABLE SOLUBILIZATION

BY TRITON THE BUOYANT

I X-100 OF ENZYMES FRACTIONS

FROM

Solubility [( % ); average (n = 6) f SD] Enzyme ol-Mannosidase Arylsulfatase C 5’-Nucleotidase Ganglioside sialidase

Control 76.0 72.7 24.5 24.4

ML

f 6.0 t

8.1

k 6.4 f 6.0

77.7 74.0 28.3 5.6

IV f f f f

2.0 6.6 7.6 2.0

Note. The recovery of the marker enzyme and the ganglioside sialidase following this treatment in both cell lines was 75-90% of the initial activity. No differences in recovery between the cell line could be observed.

GANGLIOSIDE

SIALIDASE

0

4

B

IN

12

16

MUCOLIPIDOSIS

20

24 26 Fraction

32 36 Number

TYPE

40

4

46

IV

52

FIBROBLASTS

56

605

60

FIG. 2. Distribution pattern of ganglioside sialidase in normal (A) and ML IV (B) fibroblasts on 6.7% Percoll (starting density). 0, 5’-Nucleotidase; X, arylsulfatase C; 0, ganglioside sialidase. The arrows indicate the peak activity of 1. fl-hexosaminidase, and 2. galactosyl transferase. Na+/K+ ATPase showed distribution similar to S’nucleotidase.

the 6.7% gradient was obtained by the throughout the gradient with small peaks, none of which coincided with the plasma 5.6% Percoll starting density gradient, but here the plasma membrane particles membrane markers (Fig. 3). Plasma were partia.lly separated from the micromembranes were distributed bimodally, and ganglioside sialidase was found as somes. The distribution of enzyme markers in the different gradients was consis- before to run parallel to these fragments. tent and similar in all cell lines. By these two gradients ganglioside sialidase was DISCUSSION found to run parallel to plasma membrane markers in both ML IV and control cells, It has been shown previously that ML but its distribution in the two subfractions IV cultured fibroblasts and amniotic fluid was significantly different in the two cell cells are deficient in a solubilized gangliolines. While in controls most of the gan- side sialidase (9, 6). To shed more light glioside sialidase activity was found in on the nature of this deficiency, subcellular the “lighter”’ particles, in ML IV cells this fractionations were performed by techactivity was located mainly in the heavy niques which allowed for a clear separafraction, with significantly less activity in tion of dense lysosomes from plasma the light fraction when compared to con- membrane fragments and from other cell particles. trols. The reduced activity in the lighter subfraction was variable in ML IV, so The majority, if not all, of ganglioside that in some experiments hardly any ac- sialidase in both ML IV and controls was tivity was (detected in this subfraction. associated with plasma membranes, which Identical sialidase distribution, although correlates well with what has been found lower in activity, was observed when en- in brain cells (21-25) or thyroid (29). Gansialidase activity in ML IV zyme incubations were performed in the glioside absence of Triton X-100, or when Na+ in this fraction was consistently reduced taurocholate replaced the Triton. when compared to controls as exhibited In an attempt to produce smaller plasma by the 30% Percoll gradient. An altered membrane and microsome fragments, fi- enzyme structure or altered microdistribroblasts were homogenized by 20 strokes bution was indicated by the difference in enzyme solubility. Whereas 24% of the of a piston-driven Teflon-glass homogenizer instead of nitrogen cavitation. By enzyme was solubilized by ultrasonication this procedure microsomes were dispersed in the presence of Triton X-100 in the

ZEIGLER

AND

BACH

FIG. 3. Distribution pattern of ganglioside sialidase in normal (A) and ML IV (B) fibroblasts on 6.7% Percoll (starting density). Fibroblasts were homogenized by 20 strokes of a motor-driven Teflon-glass homogenizer instead of the routine nitrogen cavitation (see text). 0, 5’-Nucleotidase; X, arylsulfatase C; 0, ganglioside sialidase. The arrows indicate the peak activity of 1. fl-hexosaminidase, and 2. galactosyl transferase.

sialidase in control fibroblasts was located pooled and concentrated buoyant fraction mainly in the lighter subfraction of the of control cells only, 6% of this activity could be solubilized in ML IV cells. The membrane, whereas in ML IV cells there solubility of 5’-nucleotidase was about 26% was a reduction in enzyme activity in this as compared to 73% of arylsulfatase C, a fraction, and enzyme activity was found microsomal marker which further indimainly in the heavy particles. Whether a cates the localization of ganglioside sialparticular isozyme is indeed deficient in idase as mostly in the plasma membrane ML IV or a shift in the localization of the in the pooled buoyant fraction. It should same isozyme occurred in the mutant cells be noted that ganglioside sialidase in total remains to be elucidated. The two plasma subfractions obviously reprecell homogenates of various ML IV pa- membrane tients exhibited identical kinetic features sent fragments of two different densities such as apparent K,, V,,,, and optimal (27). Although attempts to further charpH as the normal controls (Bach and acterize these particles were carried out Zeigler unpublished data). On 6.7 and no final conclusion on their nature could 5.6% Percoll gradients, plasma membrane be obtained. Sahagian and Neufeld (28) described fragments are separated into two distinct of fibroblasts subfractions, the nature of which was not the subcellular fractionation by 5.6% Percoll, in which the plasma entirely clarified. It is not surprising that 5’-nucleotidase was found in both light membranes were separated from other cellular organelles in a single peak. Howand heavy plasma membrane fractions, as it has been shown that 5’-nucleotidase ever, in these experiments larger fractions is situated on both the outer and the from the gradient were collected and thus the resolution of two peaks might have cytoplasmic side in cultured fibroblasts the (26). The data obtained by the latter two escaped detection and, furthermore, gradients further support the localization plasma membrane fragments were located by a lectin labeling technique and may of at least the vast majority of ganglioside sialidase in cultured fibroblasts to the represent only part of the membrane and plasma membrane in both control and not the entire membrane particles. Contrary to most lysosomal storage disML IV cells. However, there was an alorders which are usually fatal in the early tered microdistribution of enzyme activity stages of life, ML IV patients manifest in ML IV cells. Thus, the ganglioside

GANGLIOSIDE

SIALIDASE

IN

MUCOLIPIDOSIS

symptoms very early, especially that of cornea1 clouding and psychomotor retardation, but they deteriorate much more slowly. It m.ay well be that the defective ganglioside sialidase is still capable of partial ganglioside catabolism, or that other unaffected isozymes assist in the hydrolysis of gangliosides. ACKNOWLEDGMENTS The authors thank Ms. Bella Meidan for her skillful technmal assistance in propagating the cultured fibroblast.s, and Ms. Ruth Bargal for her assistance in the biochemical work. This work is part of the thesis of M.Z. for M.Sc. Part of this work was supported from the Fund for Basic Research administered by the IIsrael Academy of Sciences and Humanities, and by a grant from the Deutsche Forschungsgemeinlschaft. REFERENCES 1. BERMAN, E. R., LI~NI, N., SHAPIRA, E., MERIN, S., AND LEVIJ, I. S. (1974) J. Pediat. 84, 519526. 2. MERIN, S., LIVNI, N., BERMAN, E. R., AND YATZIV, S. (1975) Invest. Ophthalmol. 14, 437-438. 3. TELLEZ-NAVEL, I., RAPIN, I., IWAMOTO, T., JOHNSON, A. El., NORTON, W. T., AND NITOWSKY, H. (1976) AT& Neural. 33, 828-835. 4. NEWELL, F. W., MATALON, R., AND MEYERS, S. (1975) Amer. J. Ophthalmol. 80, 440-449. 5. BACH, G., COHEN, M. M., AND KOHN, G. (1975) B&hem. Biophys. Res. Commun. 66, 14831490. 6. BACH, G., ZIEIGLER, M., AND KOHN, G. (1980) Clin Chim. Acta 106, 121-128. 7. CRANDALL, B. F., PHILIPPART, M., BROWN, W. J., AND BLUE:STONE, D. (1982) Amer. J. Med Genet 12, 301-3’08. 8. BACH, G., ZEIGLER, M., KOHN, G., AND COHEN, M. M. (1’977) Amer. J. Hum. Genet. 29, 610618. 9. BACH, G., ZEIGLER, M., SCHAAP, T., AND KOEIN, G. (1979) Biochem. Biophys. Res. Commun. 90, 1341-134’7. 10. BEN-Y• SEF, Y., MOMOI, T., HAHN, L. C., AND NADLER, H. Z. (1982) Clin. Genet. 21, 374-381.

TYPE

IV

FIBROBLASTS

607

11. CAIMI, L., TE’ITAMANTI, G., BERRA, B., SALE, F. O., BORRONE, C., GATTI, R., DURAND, P., AND MARTIN, J. J. (1982) J. Inherit. Dis. 5, 218-224. 12. ROME, L. H., GARVIN, J. A., ALLIENTA, M. M., AND NEUFELD, E. F. (1979) Cell 17, 143-154. 13. ZEIGLER, M., AND BACH, G. (1981) Biochem J. 198, 505-508. 14. WARREN, L. (1959) J. Biol. Chem 234, 1971-1975. 15. VEH, R. W., AND SCHAUER, R. (1978) in Enzymes of lipid metabolism (Gatt, S., Freysz, L., and Mandel, P., eds.), Adv. Exp. Biol. Vol. 101, pp. 447-462, Plenum, New York. 16. VEH, R. W., AND SANDER, M. (1981) in Perspectives in Inherited Metabolic Diseases (Tettamanti, G., Durand, P., and DiDonato, S., eds.), Vol. 4, pp. 71-110, Edi Ermes, Milan. 17. BACH, G., AND LIEBMAN, A. (1979) Eur. J. B&hem. 96, 613-619. 18. POST, R. L., AND SEN, A. K. (1978) J. Physiol. 247, 247-263. 19. MEYER, J. CH., GRUNDMANN, H. P., AND SCHNYDER, 0. W. (1979) Arch. Dermatol. Res. 266, 95-98. 20. DALLNER, G. (1974) in Methods in Enzymology (Fleischer, S. S. and Packer, L., eds.), Vol. 31, pp. 191-201, Academic Press, New York. 21. CARUBELLI, R., TRUCCO, R. E., AND CAPUTTO, R. (1962) B&him. Biophys. Acta 60, 196-200. 22. LEIBOWITZ, Z., AND GATT, S. (1968) B&him. Biophys. Acta 152, 136-143. 23. ROUKEMA, P. A., AND HEIJKMAN, J. (1970) J. Neurochem. 17,773-780. 24. TE~AMANTI, G., PRETI, A., LOMBARDO, A., BONALI, F., AND ZAMBOTTI, V. (1973) Biochim Biophys. Acta 306, 466-477. 25. VENERANDO, B., TETTAMANTI, G., CESTARO, B., AND ZAMBOTTI, V. (1975) B&him. Biophys. Acta 403, 461-472. 26. WIDNELL, C. C., SCHNEIDER, Y. -J., PIERRE, B., BAUDHUIM, P., AND TROUET, A. (1982) Cell 28, 61-70. 27. DOMSCH, C., AND MERSMANN, G. (1982) E3cp. Cell Res. 142, 482-485. 28. SAHAGIAN, G. G., AND NEUFELD, E. F. (1983) J. Biol. Chem. 258, 7121-7128. 29. VAN DESSEL, G., DEWOLF, M., LAGROU, A., HILDERSON, H., AND DIERICK, W. (1984) J. B&hem. 96, 937-947.