Occurrence of creatine kinase activity in human erythrocyte membrane.

Some evidence for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.

Incorporation and degradation of GM1 ganglioside and asialoGM1 ganglioside in cultured fibroblasts from normal individuals and patients with beta-galactosidase deficiency.

The uptake and degradation of GM1 ganglioside (GM1) and asialoGM1 ganglioside (GA1) were studied in cultured fibroblasts from normal individuals and patients with beta-galactosidase deficiency, using the lipid-loading test. The glycolipids were incorporated from the media into the fibroblasts and the terminal galactose was hydrolyzed in normal cells. The hydrolysis rates of GA1 were 80-86% of normal on the 3rd day after loading, while GM1 was hydrolyzed slowly; 35-54% on the 14th day. In infantile GM1 gangliosidosis and I-cell disease, little GM1 and GA1 was hydrolyzed on any day of culture, while fibroblasts from patients with adult GM1 gangliosidosis, Morquio disease type B and galactosialidosis hydrolyzed the lipids at nearly normal rates. The intracellular accumulation of the glycolipids, on the basis of protein content, was abnormally high in the case of infantile GM1 gangliosidosis and I-cell disease, but normal in the other disorders examined. These observations indicate that the in situ metabolism of GM1 and GA1 is probably normal in fibroblasts from patients with adult GM1 gangliosidosis, Morquio disease type B and galactosialidosis, although in vitro beta-galactosidase activities in these disorders are very low. The results are compatible with findings that GM1 and GA1 do not accumulate in the somatic organs of patients with adult GM1 gangliosidosis and galactosialidosis. In I-cell disease, however, the results of the loading test did not agree with the finding that there is little accumulation of glycolipids in postmortem tissues.

Metabolism of galactosylceramide in the twitcher mouse, an animal model of human globoid cell leukodystrophy.

The metabolism of galactosylceramide was investigated in normal and twitcher mice, an animal model for human globoid cell leukodystrophy. The findings were compared with data obtained on human tissues. In vitro studies demonstrated that there were two genetically distinct enzymes that hydrolyze galactosylceramide: galactosylceramidase I and II. The former was deficient in the twitcher, while the latter was intact. beta-Galactosidase preparations purified from normal mouse liver possessed the activity to hydrolyze galactosylceramide when the assay conditions for galactosylceramidase II was used. Therefore, galactosylceramidase II was considered to be identical to GM1 ganglioside beta-galactosidase. In contrast to the human enzyme, the murine beta-galactosidase had a relatively high Km value toward galactosylceramide. The galactosylceramide-loading test demonstrated that the twitcher fibroblasts hydrolyzed the lipid at lower rates than seen in cases of human globoid cell leukodystrophy fibroblasts. These differences in galactosylceramidase II between murine and human tissues suggest that galactosylceramide accumulates in twitcher mice but not in humans with globoid cell leukodystrophy, even though galactosylceramidase I is genetically deficient in both human and this mouse model.

Galactosylceramide- and lactosylceramide-loading studies in cultured fibroblasts from normal individuals and patients with globoid cell leukodystrophy (Krabbe's disease) and GM1-gangliosidosis.

The metabolism of galactosylceramide and lactosylceramide in cultured fibroblasts was studied using the lipid-loading test. These compounds were incorporated into the fibroblasts yet only small amounts of the incorporated lipids were hydrolyzed unless additional phospholipid was mixed with the glycolipid before loading. Among phospholipids, phosphatidylserine was the most effective for incorporation and hydrolysis of the glycolipids, while phosphatidylcholine inhibited the incorporation of the glycolipids. Using filtration techniques, light scattering analyses and subcellular fractionation, the particle size of glycolipid in the culture medium was found to be critically important for the incorporation of the lipids into the cells and their transportation to the lysosomes. The particle sizes of the glycolipids were decreased by mixing with phosphatidylserine. Furthermore, the negative charge in phosphatidylserine may be necessary for the glycolipid transportation into the lysosomes. In fibroblasts from patients with globoid cell leukodystrophy, 40-50% of galactosylceramide was hydrolyzed on the 4th day of culture, a time when the control fibroblasts had hydrolyzed it about 80%. This finding is in contrast with observations made on fibroblasts with other sphingolipidoses which showed near-zero degradation in corresponding substrate-loading tests. In fibroblasts from patients with either globoid cell leukodystrophy of GM1-gangliosidosis, hydrolysis of lactosylceramide was fairly normal yet somewhat lower than control values on any day of culture, thereby indicating that, in the loading tests, lactosylceramide seems to be hydrolyzed with similar levels of enzyme activities by two distinct beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.

Adrenoleukodystrophy protein enhances association of very long-chain acyl-coenzyme A synthetase with the peroxisome.

OBJECTIVE: To clarify the function of adrenoleukodystrophy protein (ALDP) using our ALDP-deficient mice established by gene targeting. BACKGROUND: X-linked adrenoleukodystrophy (ALD) is characterized biochemically by the accumulation of very long-chain fatty acids (VLCFA) in tissues and body fluids, and is caused by impairment of peroxisomal beta-oxidation. In ALD, very long-chain acyl-coenzyme A synthetase (VLACS), which is necessary for peroxisomal beta-oxidation, does not function. METHODS: The ALDP-deficient mice and C57BL/6J mice were used. VLACS or ALDP were transiently expressed by lipofection in murine fibroblasts, and VLCFA beta-oxidation was assayed. Liver peroxisomes were purified by sequential centrifugations and a Nycodenz gradient centrifugation. The peroxisomal localization of VLACS was compared between the mutant and control mice using a Western blot analysis. RESULTS: Impairment of VLCFA beta-oxidation in ALDP-deficient fibroblasts was not corrected by the additional expression of VLACS alone but was by the coexpression of VLACS and ALDP. Although the tissue-specific expression of VLACS was similar in ALDP-deficient and normal mice, peroxisomal VLACS was clearly lower in ALDP-deficient than in normal mice. CONCLUSIONS: ALDP plays a role in the peroxisomal localization of VLACS, and VLACS does not function unless localized in the peroxisome.

Adult-onset Krabbe disease with homozygous T1853C mutation in the galactocerebrosidase gene. Unusual MRI findings of corticospinal tract demyelination.

A 51-year-old woman developed a slowly progressive spastic paraparesis and diminished vibration sense beginning at age 38. Intellectual capacity was normal. Krabbe disease was confirmed by markedly reduced leukocyte galactocerebrosidase (GALC) activity, typical inclusions in Schwann cell cytoplasm, and an identification of the homozygous point mutation T1835C (Leu618Ser) in the GALC gene. T2-weighted MRI of the brain showed symmetric high-signal-intensity lesions in the bilateral frontoparietal white matter, the centrum semiovale, and the posterior limb of the internal capsule with sparing of the periventricular white matter. This case is unusual because of the late onset, protracted clinical course, and MRI findings of demyelination confined to the corticospinal tracts.

Very-long-chain fatty acid metabolism in adrenoleukodystrophy protein-deficient mice.

X-linked adrenoleukodystrophy (X-ALD) is characterized by progressive mental and motor deterioration, with demyelination of the central and peripheral nervous system. Its principal biochemical abnormality is the accumulation of very-long-chain fatty acids (VLCFAs) in tissues and body fluids, caused by the impairment of peroxisomal beta-oxidation. The authors have generated a line of mice deficient in ALD protein (ALDP) by gene targeting. ALDP-deficient mice appeared normal clinically, at least up to 12 mo. Western blot analysis showed absence of ALDP in the brain, spinal cord, lung, and kidney. The amounts of C26:0 increased by 240% in the spinal cord. VLCFA beta-oxidation in cultured hepatocytes was reduced to 50% of normal. The authors investigated the roles of ALDP in VLCFA beta-oxidation using the ALDP-deficient mice. Very-long-chain acyl-CoA synthetase (VLACS) is functionally deficient in ALD cells. The impairment of VLCFA beta-oxidation in the ALDP-deficient fibroblasts was not corrected by over-expression of VLACS only, but was done by co-expression of VLACS and ALDP, suggesting that VLACS requires ALDP to function. VLACS was detected in the peroxisomal and microsomal fractions of the liver from both types of mice. Peroxisomal VLACS was clearly decreased in the ALDP-deficient mouse. Thus, ALDP is involved in the peroxisomal localization of VLACS.

Glucosylceramide and glucosylsphingosine metabolism in cultured fibroblasts deficient in acid beta-glucosidase activity.

The metabolism of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) was studied using cultured fibroblasts deficient in acid beta-glucosidase activity. In fibroblasts from patients with Gaucher's disease, in vitro beta-glucosidase activities were 2.7-11.7% and 4.8-13.6% of control values when 4-methylumbelliferyl beta-D-glucoside and GlcSph were used as substrates, respectively. In spite of the enzyme deficiency, GlcCer and GlcSph, the natural substrates of the deficient enzyme, did not accumulate in the cells. When normal fibroblasts were incubated with conduritol B epoxide (CBE), a specific inhibitor of acid beta-glucosidase, the in vitro enzyme activities decreased dose-dependently (2.2-2.4% of control values at 50 microM CBE), and GlcCer and GlcSph accumulated in the cells at concentrations of CBE higher than 50 microM. To investigate the intracellular metabolism of GlcCer and GlcSph, either radioactive GlcCer or GlcSph was loaded onto cultured fibroblasts. In fibroblasts treated with a high dose of CBE (1 mM), the degradation of GlcCer and GlcSph was retarded (5-21% on day 7; normal range, 81-99%), while in fibroblasts from patients with Gaucher's disease, both the pattern and rate of the degradation of the lipids (83-97% on day 7) were almost the same as those seen in the control cells. These results indicate that in Gaucher's disease fibroblasts the intracellular metabolism of GlcCer and GlcSph is normal in spite of the deficiency in beta-glucosidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of ceramide trihexoside in cultured skin fibroblasts from Fabry's patients, carriers and normal controls.

The metabolism of ceramide trihexoside (CTH) was studied in cultured skin fibroblasts from Fabry's patients, carriers, and controls. The in vitro activities of alpha-galactosidase in fibroblasts using either artificial substrate or CTH led to a clear identification of 4 Fabry's patients, 6 carriers and 8 controls. Normal findings were noted in one carrier. In fibroblasts from Fabry's patients, the residual activities assayed with CTH were less than those with the artificial substrate. In the CTH-loading test, the incorporation and hydrolysis of CTH in the fibroblasts were closely related with the culture time and CTH-concentration in the medium. With 0.5 nmol CTH per flask (25 cm2 growth area, 3 ml of medium), the hydrolysis rate of CTH, based on its incorporation, was 2.1% in fibroblasts from 4 Fabry's patients, 82.0% in 10 controls and 47.1% in 6 out of 7 carriers on the 7th day of culture. The impaired hydrolysis in carriers' fibroblasts differs from the hydrolysis noted in fibroblasts from carriers of autosomal recessive sphingolipidoses with a normal hydrolysis. These findings explain that some of the carriers show clinical symptoms and a mild accumulation of CTH in their tissues, such as seen in Fabry's patients. In one carrier there was a normal hydrolysis of CTH, both in vitro and in the CTH-loading study. This may be explained by Lyon's random X-chromosome inactivation theory.

Fabry's disease with partially deficient hydrolysis of ceramide trihexoside.

A report is presented on biochemical studies of the fibroblasts from a 26-year-old man with Fabry's disease whose clinical picture was atypical. The patient had severe pain in the extremities, but no angiokeratomas, corneal clouding or hypohidrosis. The trihexosylceramidase activity in the fibroblasts in vitro was deficient. The level and Km value of the residual activity were similar to levels seen in typical Fabry's patients. However, fibroblasts from the patient cultured in medium supplemented with [3H]ceramide trihexoside hydrolyzed the labeled ceramide trihexoside much higher than did cells from typical Fabry's patients, implying that the patient has a partial defect in hydrolysis of trihexosylceramide in cultured fibroblasts.

Molecular analysis of X-linked adrenoleukodystrophy patients.

A molecular analysis of 4 Japanese adrenoleukodystrophy (ALD) patients was carried out, according to the recently published report on ALD gene cDNA. In a Southern blot analysis, we were not able to detect a large deletion in all patients. In a Northern blot analysis, no mRNA was detected in one patient, while the others had normal mRNA in both size and amount. Three patients had missense mutations including; 534Pro-->Leu (1987C-->T), 660Arg-->Trp (2364C-->T), and 512Gly-->Ser (1920G-->A), respectively. These mutations existed in the C-terminal region conserved in the ATP-binding cassette superfamily of transporters. In a Western blot analysis using polyclonal antibodies against the C-terminal peptide as well as the whole peptide of ALD protein, no 80 kDa protein was found in any of the 4 patients, which was observed in the control cells. The ALD protein in 3 patients with a missense mutation might be degraded immediately after translation because of the unstable higher structure or by the disruption of the hitherto unknown targetting signal to the peroxisome. The molecular analysis of the ALD gene as done in this study is thus considered to be the first step to further elucidate the pathogenic mechanism of ALD.

A high-performance liquid chromatographic assay for acid ceramidase activity in cultured fibroblasts from patients with Farber's disease and from controls.

The high-performance liquid chromatographic (HPLC) method we devised for assay of acid ceramidase activity involves coupling of a fluorescent probe to the enzymatically released sphingosine in the reaction mixture and detection of the fluorescent sphingosine derivative by reverse-phase HPLC. Using the method, acid ceramidase activity in fibroblast homogenates was accurately assayed, with or without the addition of exogenous ceramide, as the substrate, and the patients and carriers of Farber's disease could be readily diagnosed.

Expression and processing of recombinant human galactosylceramidase.

Stable transformants of CHO cells that overexpress human galactosylceramidase (GALC) were established. The GALC within the cell consisted of 50- and 30-kDa proteins. The active GALC secreted into the culture medium in large amounts consisted of the 80-kDa precursor enzyme. We confirmed that the precursor enzyme was taken up by fibroblasts via the mannose-6-phosphate receptor and processed into the 50- and 30-kDa fragments. Fragmentation was inhibited by the lysosomotropic agents chloroquine and NH4Cl, suggesting that it occurs within the lysosome. GALC mutations identified in globoid cell leukodystrophy suppressed fragmentation. Neither the 50- or 30-kDa fragment expressed had GALC activity, indicative that the entire structure is necessary for enzyme activity and that fragments expressed separately cannot associate to form the active enzyme.

Metabolism of free sphingoid bases in murine tissues and in cultured human fibroblasts.

Metabolism of free sphingoid bases present in normal tissues [Kobayashi, T., Mitsuo, K. & Goto, I. (1988) Eur. J. Biochem. 172, 747-752] was examined in mouse tissues and in human cultured fibroblasts. Subcellular fractionation studies of the mouse liver revealed most of free sphingoid bases to be in the membrane fractions. Fibroblasts from patients with Farber's disease contained concentrations of free sphingoid bases similar to those seen in the normal fibroblasts. When L-cycloserine, a potent inhibitor of the first reaction of sphingoid base synthesis, was added to the culture medium, the concentration of free sphingoid bases in fibroblasts decreased, dose-dependently. Thus, most of the free sphingoid bases in the tissue probably do not derive from the degradation of sphingolipids but are newly synthesized. Free sphingoid bases in microsomes from the brainstem and spinal cord were acylated or glycosylated when incubated with acyl-CoA or UDP-glycoside. However, the reaction for the synthesis of ceramide was much greater and more rapid than that of glycosylsphingosine synthesis. In liver microsomes, ceramide synthesis from endogenous free sphingoid bases was observed but synthesis of glycosylsphingosine was not evident. Therefore, the main pathway of metabolism of free sphingoid bases is presumably acylation (to ceramide) but not glycosylation (to lysoglycosphingolipid).

Protease inhibitors suppress the degradation of mutant adrenoleukodystrophy proteins but do not correct impairment of very long chain fatty acid metabolism in adrenoleukodystrophy fibroblasts.

The adrenoleukodystrophy (ALD) gene product, ALD protein (ALDP), was not detected in fibroblasts from our or most other patients with ALD as determined by immunoblot or immunocytochemistry. We investigated the stability of mutant ALDP and found from pulse-chase experiments that the respective half-lives of the normal and mutant #140 (Gly512Ser) and #249 (Arg660Trp) were 72.6, 32.1 and 26.1 min, indicative that mutant ALDPs are less stable than normal ones. The mutant ALDPs were detectable in fibroblasts cultured with the protease inhibitor E-64 or leupeptin. Protease inhibitor treatment for 2 to 28 days did not affect the amount of very long chain fatty acid (VLCFA), C26:0, or VLCFA beta-oxidation activity in ALD fibroblasts. Protease inhibitors therefore suppress the degradation of ALDP but do not correct the impairment of VLCFA metabolism in ALD.

Metabolism of exogenous galactosylceramide in the twitcher mouse brain.

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-14C] stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.

Biosynthesis of galactosylsphingosine (psychosine) in the twitcher mouse.

In attempts to elucidate the origin of accumulated galactosylsphingosine in the twitcher mouse, a murine model of human globoid cell leukodystrophy (Krabbe's disease), UDP-galactose:sphingosine galactosyltransferase activity was assayed in tissues from normal and twitcher mice. Among several tissues from normal, 20 day postnatal mice, the highest galactosyltransferase activity was found in the brainstem and spinal cord, followed by cerebrum, kidney and liver, in that order. Chronologically, the enzyme activity in the central nervous tissue increased with age, reached a maximum at 25 postnatal days, and declined thereafter. In the kidney and liver, however, the activity remained much the same during development. In the twitcher mouse, developmental change in the enzyme activity was similar to that seen in control mouse, but the decrease in activity in the central nervous tissue after the 25 postnatal days was more rapid. The galactosyltransferase activity and the accumulation of galactosylsphingosine in the tissue of the twitcher mouse were closely related; where and when the enzyme activity was higher, the greater was the accumulation of galactosylsphingosine in the tissue of the twitcher mouse. These results strongly suggest that the accumulated galactosylsphingosine in the twitcher mouse is synthesized mainly by UDP-galactose:sphingosine galactosyltransferase.

Adrenoleukodystrophy protein-deficient mice represent abnormality of very long chain fatty acid metabolism.

We have generated a line of mice deficient in adrenoleukodystrophy protein (ALDP) by gene targeting in order to clarify the pathophysiology of adrenoleukodystrophy (ALD). ALDP-deficient male and female mice appeared normal clinically at least up to 12 months. Western blot analysis showed the absence of ALDP in the brain, spinal cord, lung, and kidney and normal expression of PMP70 in the liver, lung, and kidney. The amounts of C26:0 increased by 73-240% in the brain, spinal cord, lung, and kidney. beta-Oxidation of very long chain fatty acids (VLCFA) in cultured hepatocytes and fibroblasts was reduced to 35-50% of normal. Light and electron microscopy did not show demyelination in the brain, spinal cord, and peripheral nerve. Thus, the deficiency of ALDP in mice impairs the peroxisomal fatty acid beta-oxidation but does not duplicate the clinical and pathological abnormalities of the human ALD. These observations suggest that the accumulation of VLCFA alone is not sufficient to cause demyelination in the nervous system.

Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases.

Two genetically distinct acid beta-galactosidases are apparently involved in the hydrolysis of galactosylceramide in fibroblasts. These beta-galactosidases were activated by different bile salts. The classical galactosylceramidase (galactosylceramidase I, EC 3.2.1.46) was activated by sodium taurocholate, while the other galactosylceramidase (galactosylceramidase II) was activated by sodium cholate. The former was genetically lacking in globoid cell leukodystrophy (GLD) and the latter in GM1 gangliosidosis. Galactosylceramidase II cross-reacted with antibody raised against purified GM1 ganglioside beta-galactosidase (EC 3.2.1.23) from the human placenta. The purified beta-galactosidase had galactosylceramidase II activity, which was competitively inhibited by GM1 ganglioside. Thus, galactosylceramidase II seems to be identical to GM1 ganglioside beta-galactosidase and lactosylceramidase II. Galactosylceramidase II had a very low affinity for galactosylsphingosine. In the galactosylceramide-loading tests using fibroblasts from patients with GLD and GM1 gangliosidosis, both cell lines hydrolyzed the incorporated galactosylceramide, with lower rates than control fibroblasts but higher than the fibroblasts from patients with I-cell disease, in which both galactosylceramidase I and II were deficient. These results indicate that galactosylceramide is hydrolyzed by two genetically distinct beta-galactosidases and explain well that galactosylsphingosine but not galactosylceramide accumulates in the brain of patients with GLD.