Nobue Shinnoh

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.

Lovastatin does not correct the accumulation of very long-chain fatty acids in tissues of adrenoleukodystrophy protein-deficient mice

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A reductase, normalizes the very long-chain fatty acids (VLCFA) concentrations in fibroblasts and plasma from patients with X-linked adrenoleukodystrophy (X-ALD). The effects of lovastatin on the accumulation of VLCFA in tissues of adrenoleukodystrophy protein (ALDP)-deficient mice were assessed. ALDP-deficient mice were fed chow with 0.01–0.1% lovastatin for 4–8 weeks. The VLCFA concentrations in the plasma, brain, spinal cord, liver and kidneys were measured. Treatment with 0.1% lovastatin significantly reduced body weight and total cholesterol in the plasma of ALDP-deficient mice. Treatment with lovastatin, however, did not correct the accumulation of VLCFA in the plasma or tissues, including the brain and spinal cord. Lovastatin does not affect the accumulation of VLCFA in ALDP-deficient tissues in mice.

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 β-oxidation. In ALD, very long-chain acyl-coenzyme A synthetase (VLACS), which is necessary for peroxisomal β-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 β-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 β-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.

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.

Therapeutic effects of normal cells on ABCD1 deficient cells in vitro and hematopoietic cell transplantation in the X-ALD mouse model

Bone marrow transplantation (BMT) is accepted as an efficient therapy for X-linked adrenoleukodystrophy (ALD). To clarify the mechanisms of this treatment, we examined the effects of hematopoietic cell transplantation (HCT) in an ATP-binding cassette, subfamily D, member 1 (ABCD1) knock out mice and co-culture of ALD patient fibroblasts with normal cells. We treated ABCD1 knock out mice with HCT using lacZ-transgenic mice as donors, which enabled us to detect donor-derived cells. We also examined the effects of co-culturing a normal microglia cell line (N9) with ALD fibroblasts. beta-Galactosidase (beta-GAL) activity was higher in spleen, lung and kidney than in liver, brain and spinal cord of the recipient ABCD1 knock out mice. HCT reduced the accumulation of very long chain fatty acid (VLCFA) in those tissues. The reduction of the VLCFA ratio was significant in spleen and lung; tissues with higher beta-GAL activity. ABCD1 was detectable in spleen from HCT mice. Co-culture of ALD fibroblasts with normal fibroblast cells reduced VLCFA accumulation in ALD cells. This effect was not observed when the cells were co-cultured while separated by a filter membrane. Our data suggest that supplying normal cells for ABCD1 knockout mouse by HCT corrects metabolic abnormalities in ALD tissues through a cell-mediated process. The correction requires direct cell-to-cell contact for recovering normal cell function.

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 β-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 β-oxidation in cultured hepatocytes was reduced to 50% of normal. The authors investigated the roles of ALDP in VLCFA β-oxidation using the ALDP-deficient mice. Very-long-chain acyl-CoA synthetase (VLACS) is functionally deficient in ALD cells. The impairment of VLCFA β-oxidation in the ALDP-deficient fibroblasts was not corrected by overexpression 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.

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 (Krabbes 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.

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 Fabrys 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 Fabrys 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 Fabrys patients, implying that the patient has a partial defect in hydrolysis of trihexosylceramide in cultured fibroblasts.

Prenatal diagnosis of congenital sialidosis.

Sasagasako N, Miyahara S, Saito N, Shinnoh N, Kobayashi T, Goto I. Prenatal diagnosis of congenital sialidosis.

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse.

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [32P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [3H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [3H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.