Am JHum Genet 29:610-618, 1977

Mucopolysaccharide Accumulation in Cultured Skin Fibroblasts Derived from Patients with Mucolipidosis IV GIDEON BACH,1 MARCIA ZIEGLER, GERTRUDE KOHN, AND MAIMON M. COHEN INTRODUCTION

Mucolipidosis IV (ML IV), a recently described autosomal recessive trait, is characterized clinically by psychomotor retardation and bilateral congenital corneal opacities [1, 2]. Evidence that ML IV constitutes a metabolic storage disorder was obtained from electron microscopic examination of various tissues and organs of affected patients. The presence of storage bodies, similar to those found in other mucolipidoses [3], was observed in ML IV cells. Lipid-like material, manifesting a concentric lamellar configuration (membranous cytoplasmic bodies), as well as amorphous granular material, presumably water soluble, were observed. The lysosomal origin of the storage organelles in ML IV has been documented histochemically [1]. Since the description of the first patient in 1974, four additional cases have been identified in Israel [1, 2] and several more in the United States [4, 5], all of whom were of Ashkenazi Jewish origin. Elevated amounts and abnormal distribution of GM3 and GD3 gangliosides were recently reported in the cultured skin fibroblasts of several ML IV patients [6]. The present paper identifies the water soluble material accumulating in ML IV as acid mucopolysaccharides (MPS) of which hyaluronic acid (HA) is the major constituent. MATERIALS AND METHODS

Cell Culture Skin fibroblasts from three unrelated ML IV patients and two normal controls were propagated by standard techniques as previously described [6]. The patients (A, B, C) were 2-3-years-old, and the controls (strains 1 and 2) were 3- and 4-years-old at the time of biopsy. The cells were grown in nutrient mixture F-10 (HAM; Gibco, Grand Island, N.Y.) supplemented with 10% fetal calf serum in 75 cm2 Falcon tissue culture flasks in an atmosphere of 95% air and 5% Co2. Confluent monolayers of skin fibroblasts between passage levels 5-25, containing roughly 5 X 106 cells per culture flask (approximately 1 mg protein), were used for all investigations. Unless stated otherwise, all of the following experiments were performed on five repeated determinations (one culture flask each) of the three ML IV and both control cell strains. Since the within strain replication was quite consistent and between strain variability (both ML IV and controls) minimal, all results are presented as the mean and standard deviation of multiple determinations for each cell type. Received August 23, 1976; revised May 25, 1977. This work was supported by grant no. 371 from the Israel Ministry of Health. M. M. Cohen is an Established Investigator of the Chief Scientist's Bureau, Israel Ministry of Health. 1 All authors: Department of Human Genetics, Hadassah-Hebrew University Medical School, Jerusalem, Israel. © 1977 by the American Society of Human Genetics. All rights reserved.

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Incorporation of Radioactive Precursors Intracellular materials were labeled with tritiated glucose, galactose, or glucosamine(lpuCi/ ml) [Radiochemical Center, Amersham, England]. The isotope was supplied in glucose-free medium supplemented with 2% glucose. Labeling with 35S-sulfate (Radiochemical Center) was carried out in the same medium (10 ,uCi/ml). These isotopic concentrations were used in all studies. Cells were incubated with the appropriate radioactive precursor for 48 hr at 370C in an atmosphere of 95% air and 5% C02, prior to extraction and biochemical determinations. Mucopolysaccharide (MPS) Extraction Lipids were extracted by chloroform-methanol treatment as previously described [6]. The

lipid-free residue was then further extracted into 2 ml of 2.5 M NaCl with a teflon hand homogenizer, followed by centrifugation at 12,000 g for 20 min. The pellet was incubated overnight with 300 Ag Pronase (Calbiochem, Monsey, N.Y.) at 30'C and pH 6.8 in a total volume of 2 ml of 0.1 M sodium phosphate buffer. The undigested material was removed by centrifugation and the supernatant combined with the 2.5 M NaCl extract. DEAE-Sephadex Chromatography Cell extracts, prepared as above, containing approximately 130,000 cpm of either 3H labeled glucosamine, glucose, or galactose per mg protein (one culture flask) were dialyzed overnight against 0.005 M phosphate buffer at pH 7.0 and then applied to a 1 x 10 cm DEAE-Sephadex column (A-50, Pharmacia, Uppsala, Sweden), previously equilibrated against the buffer. Stepwise elution, with 50 ml fractions of increasing NaCl concentrations, was performed as described by Bach and Berman [7]. Eluted fractions were dialyzed overnight against water, concentrated to 1 ml by lyophilization, and their radioactivity determined in 10 ml Insta Gel

(Packard, Downers Grove, Ill.). Cellulose Acetate Electrophoresis Approximately 10-20 Ag MPS, as determined by uronic acid analysis [8], were applied to the cathodal end of a 2.5 x 16.75 cm cellulose acetate strip (Sepharose III, Gelman, Ann Arbor, Mich.). In experiments using labeled MPS, nonradioactive standards were added as carriers. Electrophoresis, performed in 0. 1 M barbital buffer (pH 8.6) for 40 min at 200 V (constant voltage) and 4°C, separated heparan sulfate (from E. Berman, Jerusalem), hyaluronic acid, chondroitin 4- and 6-sulfate, and dermatan sulfate (Miles Laboratories, Elkhart, Ind.). MPS was located and extracted from the strips as described by Endo and Yosizawa [9]. Determination of radioactively labeled MPS was achieved by serially counting 1 cm pieces of the strip placed in

H20 and 10 ml Insta Gel. Accumulation of 35SO4 Containing Compounds 0.5 ml

The accumulation of 35S-labeled macromolecules was investigated by the technique of Fratantoni et al. [10]. Cells were incubated with 15 x 106 cpm H235S04 per ml. At intervals of 1, 2, 3, 5, and 7 days, the cells were washed with isotonic saline, trypsinized, rewashed twice, and extracted in boiling 80% ethanol four times. Cell residues were dissolved in warm 10% NaOH (w/v) followed by determination of radioactivity and protein concentration. For these studies, five repeat experiments using ML IV strains A + B and the two control strains were performed. Pulse-Chase Studies The accumulation and disappearance of HA and sulfated MPS were investigated by incubating for both 2 and 5 days with the appropriate precursor (3H-glucosamine, 1 uCi/ml and 35S-sulfate, 10 ,uCi/ml, respectively), replacing the radioactive medium with fresh "cold" medium and harvesting the cells by trypsinization at 6, 24, 48, 72, and 96 hr. The spent medium from each flask was diluted with 4 vol of absolute ethanol, centrifuged, and the radioactivity of resulting pellet (containing mainly water soluble material), dissolved in 1 ml distilled water, was determined. Intracellular MPS was extracted as described above. Following dialysis, these

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cellular extracts were subjected to cellulose acetate electrophoresis. For these studies, four repeat experiments were performed using ML IV strain A and control strain 1.

Chemical Analyses Total hexosamine was assayed by a modification [11] of the Elson-Morgan reaction [12]; uronic acid according to Bitter and Muir [8]; sulfate by the method of Dodgson et al. [13]; and total protein by the Lowry technique [14]. Overnight incubation of MPS with hyaluronidase (Streptomyces hyalurolyticus) from Calbiochem was carried out in 0.1 M acetate buffer at pH 5.0 and 50'C [15]. MPS extract (10 pg) was incubated with 15 turbidity reducing units (TRU) overnight, dialyzed against water, and concentrated to minimal volume, followed by cellulose acetate electrophoresis. Three repeat experiments using ML IV strain A and control strain 1 were

performed. RESULTS

Distribution of Labeled Materials There were no apparent differences in radioactivity between ML IV and normal fibroblasts in the total water soluble extract, regardless of the isotope used. However, significant differences between the radioactively labeled compounds were observed following DEAE-Sephadex chromatography. Similar elution patterns for the intracellular materials were obtained by using either isotopic glucosamine, glucose, or galactose. Figure 1 presents the mean results of chromatography of three ML IV and two control cell strains after a 48 hr exposure to 3H-glucosamine (1 gCi/ml) prior to harvest and extraction. In normal cells (fig. lB), most of the radioactivity was eluted by salt concentrations up to 0.2 M. In the ML IV cell extracts, significant amounts of radioactivity were detected in the higher salt concentration fractions (fig. IA). The control cells contained 15% of their total radioactivity in fractions 5-7 and the ML IV cells, 41%. These latter fractions normally contain acid mucopolysaccharides while the former include principally neutral or slightly acidic glycoproteins [7].

Electrophoresis Since DEAE-Sephadex chromatography does not separate the various types of MPS, elution fractions 5-7 (fig. 1) were combined and subjected to cellulose acetate electrophoresis. Figure 2A indicates a marked increase of labeled HA and sulfated MPS (heparan sulfate and dermatan sulfate plus chondroitin sulfate) in all three ML IV fibroblast strains following exposure to 3H-glucosamine. While intracellular HA reached 30%-40% of the total MPS in the two normal cell strains (fig. 2B), it comprised approximately 70% of MPS in all three ML IV cell strains (fig. 2A), which is a 10-fold increase. Chemical Determinations Chemical analysis of the HA and sulfated MPS separated by cellulose acetate electrophoresis demonstrated similar increases in the MPS content of ML IV fibroblasts. This procedure was found to recover approximately 50% of the MPS labeled with 3H-glucosamine. Analysis of the uronic acid, hexosamine, and sulfate content of both extracted HA and sulfated MPS demonstrated the expected theoretical molar ratios with no significant differences between ML IV and control cells. Slight

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oversulfation was observed in sulfated MPS, but the HA was sulfate free. Table 1 indicates that in all three ML IV strains the total HA content was increased approximately 10-fold and sulfated MPS three times the control values; data compatible with those of the isotopic labeling experiments (fig. 2). Confirmation that HA was accumulating in the cells is indicated by an 80% decrease of HA content following incubation of glucosamine labeled cell extracts with fungal hyaluronidase with no changes in sulfated MPS. Accumulation Studies Figure 3 demonstrates the accumulation of sulfated MPS in ML IV cultured

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fibroblasts. Increasing linear accumulation of 35S-sulfated compounds throughout the culture period was observed in both ML IV strains. In contrast, normal fibroblasts maintained a steady state after 48 hr. Pulse-Chase Experiments "Pulse-chase" experiments reveal a much slower release of sulfated MPS and HA TABLE 1 FROM CELLULOSE ACETATE STRIPS EXTRACTED MPS OF ANALYSIS QUANTITATIVE Hyaluronic Acid* Cell Strain

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from ML IV fibroblasts (fig. 4). After only 2 days of prelabeling, marked differences were noted in the rate of disappearance of HA in the ML IV and control fibroblasts (fig. 4A). On the other hand, after 2 days of incubation, sulfated MPS disappearance was similar in both cell types. Significant differences in sulfated MPS disappearance became obvious only after 5 days of incubation with the precursor before the "chase" (fig. 4B). However, kinetics of HA disappearance did not change appreciably after 5 days of preincubation. The control cells behaved similarly regardless of the precursor used or the length of preincubation. Disappearance rates of HA and sulfated MPS in the ML IV cells differed considerably; the latter decreasing faster than HA. After a 24 hr "chase," 50% of the sulfated MPS remained compared to 90% of HA. At 48 hr, only 35% of sulfated MPS remained with no change in HA content. Over 80% of the radioactivity recovered from the medium of either normal or ML IV fibroblasts during the chase was not precipitable by ethanol but was dialyzable, indicating the presence of low molecular weight fragments. DISCUSSION

ML IV was first described 3 years ago [1], but present knowledge still mainly consists of its clinical manifestations and the ultrastructural changes observed in various organs of affected patients. Although additional cases have been studied [2, 4, 5], the basic defect remains unidentified, and biochemical efforts have concentrated on

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the identification of the accumulation products. Increased amounts of both water soluble and lipid-like storage material [1, 2], as demonstrated by electron microscopy, indicate that ML IV is a mucolipidosis. Identical findings in cultured amniotic fluid cells of affected fetuses have facilitated the prenatal diagnosis of ML IV [16]. Recently, GM3 and GD3 gangliosides were identified as the major constituents of the accumulated lipid-like material in cultured ML IV skin fibroblasts [6]. Similarly, increased levels of gangliosides have been found in the brain biopsy of a 7-year-old patient [5], an observation which not only supports our earlier findings but confirms in vivo the in vitro studies. Acid mucopolysaccharides comprise the major water soluble polymeric carbohydrate-containing constituent that accumulates in cultured ML IV fibroblasts. However,

MUCOPOLYSACCHARIDE ACCUMULATION IN ML IV 617 the techniques used in this study would not detect storage of unglycosylated proteins or small, dialyzable fragments. DEAE-Sephadex chromatography demonstrated no abnormal increases in the neutral or slightly acidic glycoprotein fractions. Both direct chemical analysis as well as the experiments using radioactive labeled precursors indicate an increase of HA (10-fold) and sulfated MPS (threefold) in ML IV fibroblasts. As a consequence, HA comprised 70% of total MPS in ML IV compared to 30% in controls. No apparent structural or electrophoretic mobility differences were observed in MPS extracted from either cell type. Such accumulation may result from either excess biosynthesis or faulty catabolism. "Pulse-chase" experiments (fig. 4) strongly suggest that the degradation process is defective. It should be noted that the accumulation kinetics of 35S-labeled macromolecules in ML IV fibroblasts is different from that in the mucopolysaccharidoses [10]. In the latter, marked accumulation of sulfated macromolecules becomes obvious after 2 days of incubation while in ML IV, similar differences were noted only after 4-5 days. Similarly, the "chase" of MPS molecules in ML IV necessitated 5 days of labeling as opposed to 2 days in the mucopolysaccharidoses [10]. These observations may indicate that the sulfated MPS may be stored in different cellular pools in the two conditions. HA, however, is probably stored in a different pool from the sulfated MPS since both its accumulation and disappearance from the ML IV cells differs noticeably from normal individuals after only 2 days of labeling. Ineffective catabolism, resulting from the mutation of a lysosomal hydrolase, may be a primary defect as in other storage disorders [17]. It may also be secondary to the inhibition of one or more of the hydrolases by other substances accumulating in the lysosome. However, all the lysosomal enzymes thus far investigated in ML IV cells have been normal [1, 5]. The previous finding of increased gangliosides in ML IV [6] cannot, as yet, be related to the present data. However, it may suggest the deficiency of an enzyme which is necessary for the catabolism of both classes of accumulated material or that their degradation is interdependent. The simultaneous storage of both glycolipids and MPS excludes the possibility of yet another mucopolysaccharidosis, and clearly characterizes this disease as a mucolipidosis [13]. In fact, accumulation of acidic MPS has been demonstrated in the cultured fibroblasts obtained from other mucolipidoses, namely ML II and ML III [18, 19]. SUMMARY

Increased concentrations of total sulfated mucopolysaccharides (MPS), threefold, and hyaluronic acid (HA), 10-fold, were found in ML IV fibroblast extracts when compared to normal controls. Such accumulations altered the distribution of MPS: HA comprised 70% of total MPS in ML IV but only 30% in control cells. Intracellular sulfated MPS was observed accumulating almost linearly in ML IV fibroblasts. "Pulse-chase" experiments indicate that both HA and the sulfated MPS remain in the ML IV cells for long periods of time; in control cells, they are rapidly removed as low molecular weight, dialyzable fragments. These data suggest that the MPS accumulation in ML IV fibroblasts, is the consequence of a catabolic block, probably involving the lysosome.

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