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Alterations in Cultured Fibroblasts of Sibs with an Infantile Form of a Free (Unbound) Sialic Acid Storage Disorder

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Alterations in Cultured Fibroblasts of Sibs with an Infantile Form of a Free (Unbound) Sialic Acid Storage Disorder Pediatr. Res. 17: 307-312 (1983) Alterations in Cultured Fibroblasts of Sibs with an Infantile Form of a Free (Unbound) Sialic Acid Storage Disorder GEORGE H. THO MAS,'^^' JANE SCOCCA, JACQUES LIBERT, ESTER VAMOS, CAROL S. MILLER, AND LINDA W. REYNOLDS The John F. Kennedy Institute, Baltimore, Maryland [G.H.T., J.S., C.S.M., L. W.R.]; Department of Pediatrics and Medicine, The Johns Hopkins University, School of Medicine, Baltimore, Maryland [G.H. T.]; Department of Ophthalmology, Hospital Saint Pierre, UniversitP Libre de Bruxelles, Brussels, Belgium [J.L.]; and Department of Pediatrics, Hospital Saint-Pierre, Universitd Libre de Bruxelles, Brussels, Belgium [E. V.] Summary storage of "bound" sialic acid but, rather, from an increased accumulation and excretion of free sialic acid. In contrast to the Cultured fibroblasts from two sibs with generalized hypertonia, sialidosis patients, these individuals have normal to above normal hepatosplenomegaly, and psychomotor retardation within the first levels of sialidase. The clinical and/or biochemical findings in year of life were found to have unusual morphologic features. these patients clearly indicate that they suffer from a disorder When examined by phase microscopy, the unstained and unfixed distinct from either Salla disease (3, 17) or sialuria (15), both of cells contained a large number of vacuolated structures whose which are also characterized by an increased accumulation and/ gross appearance resembled that of a honeycomb in the cell or excretion of unbound sialic acid. These sibs, however, do cytoplasm. Electron microscopy studies, following fixation, showed appear to have certain features in common with patients recently the "honeycombing" to be the result of numerous, closely packed, described by Hancock et al. (5) and by Stevenson et al. (18). cytoplasmic, membrane-bound vacuoles. In some of these struc- tures the remains of fibrillogranular material could be detected. MATERIALS AND METHODS Biochemical analysis of crude snnicates of these cells revealed increased levels (4-7 x N) of an acid soluble component that Cultured fibroblasts were established from skin biopsies of the reacted with thiobarbituric acid. Analysis of trimethylsilyl deriva- patients and controls as previously described (13, 19). For bio- tives of this material by gas liquid chromatography and mass chemical studies, cells from two confluent cultures (75 cm2 tissue spectrometry showed it to be indistinguishable from sialic acid (N- culture flasks) containing approximately 2 mg protein were har- acetylneuraminic acid). vested with trypsin 7 days after subculture, pooled and washed Quantitation of this material from the cells of one of the sibs three times in phosphate buffered saline with centrifugation after after isolation on a Dowex column yielded 39.8 nmoles of free each wash step. The washed cells were resuspended in 2.0 ml of (unbound) sialic acid per mg protein whereas normal fibroblasts deionized water and ruptured by ultrasonication (three 10-sec had 1-2 nmoles per mg. Bound sialic acid levels were at the upper treatments with intervals for cooling in ice water). Free sialic acid limits of normal (24.8 versus 11-23 nmoles per mg protein). The levels in 0.1-ml samples of the sonicates were measured by the concentration of cytidine monophosphate-sialic acid was normal. thiobarbituric acid method as described by Aminoff (2) and After incubation of the patient's fibroblasts with [3H]-~-acetyl- modified by Uchida et al. (24). CMP-sialic acid was measured maunosamine for 72 h, there was a 7-fold increase (compared to after destruction of free sialic acid with sodium borohydride normal fibroblasts) in the amount of radioactivity in free sialic acid according to the method of Kean and Roseman (10). present in the acid soluble fraction. The amount of labeled, bound Where indicated, the free sialic acid was separated from crude sialic acid in the acid-insoluble pool, however, was the same in cell sonicates before quantitation and/or preparation of deriva- both patient and control fibroblasts. tives for gas liquid chromatography-mass Geckometry. For these studies the crude sonicates were treated with ~erchloricacid (final concentration of 3.5%), allowed to stand 10 min, centrifuged (7700 Abbreviations x g for 10 min) and the supernatants removed and saved for CMP, cytidine monophosphate analysis. Precipitates were washed twice with 0.2 ml of HzO and NANA, N-acetyl-neuraminic acid the washes were added to the supernatants. Supernatants were PBS, phosphate buffered saline neutralized with KOH, centrifuged to remove potassium perchlo- TCA, trichloracetic acid rate, and applied to 0.5 x 5 cm columns of Dowex 1 x 8 acetate UDP, uridine diphosphate (100-200 mesh). Two 0.5-ml water washes of the sample tube were added to the column. The columns were then eluted with 10% acetic acid (1 1). The first 5 ml of the column effluent was Over the past few years several clinical forms of sialidosis have discarded; the next 12.5 ml was collected, evaporated to dryness been described (14). Common to each of these related disorders under reduced pressure on a rotary evaporator and dissolved in has been an increased accumulation and/or excretion of sialic 1.0 ml of water for quantitation with thiobarbituric acid and/or acid (N-acetylneuraminic acid) covalently linked to a variety of preparation of derivatives. Methylesters were prepared using oligosaccharides and/or glycoproteins. Additionally, it has been methanol and Dowex 50 x 8 H+ (12). Trimethylsilylation of the demonstrated that these alterations are associated with, and pre- methyl esters of the sialic acid in the isolated samples was per- sumptively the result of, a deficiency of a lysosomal sialidase formed as described by Kamerling et al. (9). The derivatives were (neuraminidase) (4, 20, 22). analyzed by gas chromatography using a Packard Model 421 We now describe the morphologic and biochemical features of Becker gas chromatograph with a flame ionization detector, hav- fibroblasts cultured from two infants (sibs) who suffer not from a ing a 6 ft x 4 mm internal diameter column packed with 3% SE 30 308 THOMAS ET AL. at a nitrogen carrier flow rate of 40 ml per min. The temperature NaHC03 (KC Biological Inc., Lenexa, KS) supplemented with was programmed to rise from 180" to 250°C at 5OC per min. Gas heat inactivated, dialyzed fetal calf serum (13%), glutamine (1.7 chromatography-mass spectrometry analyses were performed on pmoie per ml), glucose (86 pg per ml) and 22.8 mCi per mmole a Dupont DP -102 gas chromatography unit using a 5 ft x 2 mm [3~]~-acetyl-~-mannosamine(50 pCi per ml). The cells were internal diameter column packed with 3% OV-101 at a nitrogen maintained at 37°C in a humidified atmosphere of 5% C02-95% carrier flow rate of 22 ml per min with the temperature pro- air. grammed to increase from 180°C to 280°C in 15 min (9). After incubation, the media was removed, the cells were washed To examine the synthesis of sialic acid from [3H]~-acetylman- 6 times with 1 ml quantities of PBS and harvested with trypsin nosamine, skin fibroblasts were plated in 60 mm tissue culture (13, 19). The trypsinized cells were washed 3 times with 2 ml dishes and incubated as previously described (13, 19). When quantities of PBS, suspended in 0.5 ml water and sonicated for confluent (usually 4 days), the media was removed, the cells three 10-sec periods. To 0.4 ml cell sonicates, UDP-N-acetylglu- washed with 2 ml of sterile PBS and then incubated with 2 ml of cosamine (200 nmoles), sialic acid (3 15 nmoles) and N-acetylman- glucose-free MEM/Earle7s Base buffered with 25 mM Hepes and nosamine (464 nmoles) were added, bringing the volume to 0.6 ml. Cold 25% TCA (0.4 ml) was added. After standing for 15 min on ice, the sample was centrifuged in the cold at 1084 x g for 10 Table 1. Lysosomal enzyme activities in sonicates of cultured min, and the supernatant was removed and saved. The precipitate fibroblasts of affected sibs and controls' was washed with 1 ml of cold 10% TCA and the wash combined Normal Normal with the original TCA supernatant. The TCA insoluble precipitate Enzyme Sib I Sib 2 mean + 2 S.D. range was stored at -60°C for later studies whereas the combined TCA soluble fraction was extracted with ether, neutralized with am- f PNP N-acetyl-/3-glucos- 10,785 10,823 4383 1392 3071-5742 monium hydroxide, and stored as above. arninidase'" The neutralized, soluble fractions were applied to ion-exchange PNP a-fucosidase 130 72 35 +22 18-65 columns (0.5 x 4 cm) of Dowex 1 x 8 (200-400 mesh) in the PNP P-galactosidase 810 1040 581 +I86 419-760 formate form prepared according to the method of Hurlbert et al. PNP a-mannosidase 229 469 101 +70 65-221 (8). Columns were washed overnight with deionized water before PNP P-rnannosidase 275 565 188 +I38 102-238 sample application. Radioactive metabolites were eluted and col- Aspartylglucosamini- 248 277 57 +42 27-131 lected in 0.5-ml fractions at a rate of approximately 0.5 ml per dase min according to a modification of the method of Akamatsu et al. 4Mu-N-acetyl-/3-glucos- (I) as follows: 7.5 ml of sample + water; 7.5 ml of 0.01 M formic arninidase2 acid; 7.5 ml of 1 M formic acid; 7.5 ml of 4.0 M formic acid and Total 7424 8428 3121 +I462 1966-4086 7.5 ml of 4 M formic acid/0.4 M ammonium formate. Fifty pliters %A 51 31 54 +I0 49-65 from each fraction was added to Liquiscint (National Diagnostics, ' Values expressed as nmoles of substrate cleaved per rng protein per h.
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