Immunocytochemical detection of accumulated substrates in cultured fibroblasts from patients with the infantile and adult forms of Sandhoff disease.
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Adult↗
Biomedical subjects
H Sakuraba
Publications and source records attributed to H Sakuraba.
Generation and characterization of mouse monoclonal antibodies specific for N-linked neutral oligosaccharides of glycoproteins.
We generated four monoclonal antibodies (MAbs) specific for asparagine-linked neutral oligosaccharides of glycoproteins by immunizing mice with neoglycolipids, which were derived from glycoproteins by conjugation to phosphatidylethanolamine dipalmitoyl. The binding specificity of these MAbs was determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. The four MAbs designated OMB3, OMB4, OMR5, and OMR6 reacted strongly with the neoglycolipids, Gal beta1-4GlcNAc beta1-2Man alpha1-6(Gal beta1-4GlcNAc beta1-2Man alpha1-3)Man beta1-4GlcNAc-PD, GlcNAc beta1-2Man alpha1-6(GlcNAc beta1-2Man alpha1-3)(GlcNAc beta1-4)Man beta1-4GlcNAc beta1-4GlcNAc-PD, Man alpha1-6Man beta1-4GlcNAc beta1-4(Fuc alpha1-6)GlcNAc-PD, and Man alpha1-3Man beta1-4GlcNAc-PD, respectively, that were used as immunogens. All of these MAbs exhibited a high binding specificity. The epitopes of the MAbs OMB3 and OMB4 were suggested to be nonreducing terminal trisaccharides, Gal beta1-4GlcNAc beta1-2Man-, and nonreducing beta-GlcNAc residues, respectively. MAbs OMR5 and OMR6 showed a highly restricted binding specificity, reacting only with the immunizing neoglycolipids. Subsequently, MAbs OMB3 and OMB4 were shown to react strongly with asialo-alpha1-acid-glycoprotein and asialo-agalacto-alpha1-acid-glycoprotein, respectively, by Western blotting. Furthermore, it was shown that these MAbs reacted specifically with the epitope on Chinese hamster ovary cells by an immunofluorescence technique. MAb OMB4 was also shown to detect the accumulated oligosaccharides with nonreducing terminal beta-GlcNAc residues as granular inclusions in the cultured fibroblasts from a classical Sandhoff disease patient.
Animals↗
Screening and detection of gene mutations in Japanese patients with Fabry disease by non-radioactive single-stranded conformation polymorphism analysis.
We have applied non-radioactive polymerase chain reaction (PCR)-single-stranded conformation polymorphism (SSCP) to the detection of gene mutations causing Fabry disease. Nineteen of 22 known mutations were detected as electrophoretic mobility shifts on PCR-SSCP analysis. Then, DNA from newly diagnosed Japanese patients with the classical form of Fabry disease was subjected to PCR-SSCP analysis, and 4 novel mutations (1 small deletion, 1 nonsense mutation and 2 missense mutations) and 1 neutral polymorphism were identified. Furthermore, identification of an asymptomatic heterozygote and a hemizygote with moderate clinical manifestations was successfully achieved by application of this method to a family with the variant form of Fabry disease. PCR-SSCP is useful for the gene diagnosis of etiologically heterogeneous Fabry disease.
Adolescent↗
Molecular form and subcellular distribution of acid beta-galactosidase in fibroblasts from patients with GM1 gangliosidosis, Morquio B disease and galactosialidosis.
The molecular form and subcellular distribution of acid beta-galactosidase in cultured fibroblasts from patients with beta-galactosidase deficiency (GM1-gangliosidosis, Morquio B disease and galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In type 2 galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with GM1 gangliosidosis, expressing no beta-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant beta-galactosidase.
Antibody Specificity↗
High incidence of thrombosis in Fabry's disease.
Although a high incidence of thrombotic accidents in Fabry's disease has been postulated, few investigations have been performed. To clarify the incidence of thrombosis in Fabry's disease, we undertook a systematic study on thrombosis in patients with Fabry's disease including hemizygous males and heterozygous females. Sixty patients with Fabry's disease (45 hemizygotes and 15 heterozygotes) from 36 Japanese families were subjected to clinical, biochemical and genetic investigations. We found that seven patients with Fabry's disease (4 hemizygous males and 3 heterozygous females) had experienced thrombotic accidents. Six of these thrombotic patients developed brain infarctions, including one man who had the complication of recurrent thrombophlebitis. The remaining woman showed central retinal artery occlusion and thrombophlebitis. We demonstrated a high incidence of thrombosis in Fabry's disease. Thrombotic accidents occurred not only in hemizygous males but also in heterozygous females. The complication of thrombotic accidents should be taken into account in patients with Fabry's disease.
Adult↗
Subclinical Fabry's disease occurring in the context of IgA nephropathy.
A 28-year-old male patient with both IgA nephropathy and an unusual case of Fabry's disease has been followed for 10 years. Diagnosis of both these diseases was made by histological examination of renal biopsy tissues and the enzyme activities of alpha-galactosidase A. Serial biopsies revealed the hithertofore unrecognized process of glomerular glycolipid accumulation peculiar to Fabry's disease at the initial stages of the disease. Physical examinations and routine laboratory analyses failed to show significant signs of Fabry's disease throughout the 10-year period. While alpha-galactosidase A activity is markedly decreased in the plasma of this patient as in classical Fabry hemizygotes, the activity in leukocytes and culture fibroblasts showed a considerable residual activity. Fabry's disease associated with IgA nephropathy apparently is extremely rare, and the present subclinical case is unique in that the early stages of substrate accumulation are demonstrable.
Adult↗
[alpha-Galactosidase gene mutation and its expression product in Fabry disease (alpha-galactosidase deficiency)].
Fabry disease is characterized by a deficiency of lysosomal alpha-galactosidase (alpha-Gal) and the accumulation of glycosphingolipid (e.g. predominantly globotriaosylceramide) in various tissues, mainly in lysosomes of the vascular endothelium. This disorder is currently classified into two clinical phenotypes; classical severe type and atypical variant type. Classical form patients, with clinical manifestations of generalized angiopathy of early onset, usually show no detectable alpha-Gal activity. Recently, there are also atypical form patients with residual alpha-Gal activity and late-onset cardiomyopathy without other systemic manifestations. So far, we identified a number of alpha-Gal gene mutations including partial gene deletions, splicing mutations, nonsense mutations and missense mutations. They were heterogeneous and more than half of them were missense mutations. To clarify the molecular mechanism causing the enzyme defect in the patient, various missense mutations were expressed in COS-1 cells. At least, two groups have been identified; one expressing a mutant enzyme without catalytic activity (non-functional type), and the other expressing catalytically active but unstable mutant enzyme (fragile type). The fragile type mutants were widely present in the different clinical phenotypes from classical severe type to atypical milder type including subclinical Fabry hemizygote, and the mutant enzymes were posttranslationally inactivated and degraded in the cells. The inactivation and degradation were prevented by the addition of substrate analogue; galactose or melibiose. These findings provided us with significant informations on the molecular pathology of the enzyme defect in Fabry disease, and suggested the possibility of a new therapeutic approach for this disease.
Fabry Disease↗
Metabolism of glyoxylate, the end product of purin degradation, in liver peroxisomes of fresh water fish.
In marine fish liver, degradative enzymes able to convert purines to urate have been shown to be located in the cytosol and degradative enzymes able to convert urate to urea and glyoxylate in the peroxisomes. The end products of purine degradation are urea and glyoxylate in fish. Glyoxylate may be converted to glycine by alanine:glyoxylate aminotransferase for the reutilization of purine carbons. The present report describes that hepatic alanine:glyoxylate aminotransferase is located both in the peroxisomes and in the mitochondria in fresh water fish, showing that the intracellular localization of the enzyme differs between fresh water fish and marine fish. This is the first report on the presence of alanine:glyoxylate aminotransferase in fish peroxisomes.
Acid Phosphatase↗
Only sphingolipid activator protein B (SAP-B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal alpha-galactosidase in a detergent-free liposomal system.
The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.
Animals↗
Immunofluorescence analysis of trihexosylceramide accumulated in the hearts of variant hemizygotes and heterozygotes with Fabry disease.
An immunofluorescence method was applied to detect trihexosylceramide accumulated in the cardiac tissues from a variant hemizygote and a heterozygous female with Fabry disease, the incidence of which had been suspected to be high.
Aged↗
A human protective protein gene partially overlaps the gene encoding phospholipid transfer protein on the complementary strand of DNA.
The entire human protective protein gene has been cloned, and structural analysis revealed that the gene spans 7.5kb and comprises 15 exons. Furthermore, it partially overlaps on the opposite strand with the gene encoding phospholipid transfer protein. This region of DNA on chromosome 20 appears to encode two distinct mRNAs expressing defined functional products, and the mRNAs overlap by 58 nucleotides at their 3'-untranslated ends.
Alternative Splicing↗
Aggregation of the inactive form of human alpha-galactosidase in the endoplasmic reticulum.
35S-Labeled mutant alpha-galactosidases (Q279E and R301Q) expressed in COS1 cells were detected as two forms (46-kDa protein and gel-top aggregate) on SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The 46-kDa protein disappeared rapidly, but the aggregate decreased slowly. The accumulation of aggregate was observed in COS1 cells expressing either Q279E or R301Q on Western blot analysis. The aggregate was mainly recovered in the fraction extracted with 1% Triton X-100 and had no catalytic activity. The COS1 cells expressing Q279E were treated with 10 microgram/ml brefeldin A or 5 microM monensin. Treatment with brefeldin A caused a decrease in the alpha-galactosidase activity and an increase in the amount of aggregate, but the amount of aggregate did not change on monensin treatment.
Animals↗
A new mutation (LIPA Tyr22X) of lysosomal acid lipase gene in a Japanese patient with Wolman disease.
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Anemia↗
Two novel mutations in the alpha-galactosidase gene in Japanese classical hemizygotes with Fabry disease.
Four alpha-galactosidase gene mutations were identified in Japanese male patients with Fabry disease who had no detectable alpha-galactosidase activity. Two of them were novel mutations, an 11-bp deletion in exon 2 and a g-1 to t substitution at the 3' end of the splice acceptor site in intron 1. The former caused a frameshift and led to the creation of a new stop codon at codon 118. The latter was predicted to provoke aberrant mRNA splicing followed by accelerated degradation of the mRNA. A nonsense mutation, R301X, and a 2-bp deletion starting at nucleotide position 718, which were reported previously, were also identified in unrelated patients.
Actins↗
Two novel gene mutations (Glu174-->Lys, Phe383-->Tyr) causing the "hepatic" form of carnitine palmitoyltransferase II deficiency.
Carnitine palmitoyltransferase II (CPT II) deficiency has two different clinical forms, one with "hepatic" and the other with "muscular" symptoms. We studied the molecular basis of the "hepatic" form in two Japanese siblings. Their CPT II activity in lymphoblasts was reduced to 3% of the level observed in normal controls. cDNA analysis showed that the proband was a compound heterozygote. One allele carried a new mutation, G621-->A (Glu174-->Lys). The other carried three single-base substitutions; a new mutation, T1249-->A (Phe383-->Tyr), and two previously reported polymorphisms. The brother had the same four substitutions. Neither of the two new mutations in this study was detected in the 60 alleles of 30 Japanese control subjects. Secondary structure prediction analysis of the mutated CPT II protein was different from that of the normal protein. We concluded that these mutations caused the "hepatic" form of CPT II deficiency in the probands.
Alleles↗
Coexistence of gene mutations causing Fabry disease and Duchenne muscular dystrophy in a Japanese boy.
Both Fabry disease and Duchenne muscular dystrophy were confirmed by gene analysis in a Japanese boy. He developed muscle weakness at 4 years of age. A muscle biopsy revealed lamellar inclusion bodies in vascular endothelial cells in addition to myopathic changes with negative dystrophin staining. The myopathic symptoms progressed, and he died of pneumonia at 24 years of age. No clinical manifestations of Fabry disease were observed except for hypohidrosis and angiokeratoma. However, glycolipid accumulation was found in biopsied renal tissue. Molecular analysis demonstrated two gene mutations; a novel single-base deletion in exon 3 of the alpha-galactosidase gene, and a dystrophin gene deletion extending from exon 46 to exon 50. His mother was confirmed to be heterozygous for both gene deletions.
Adult↗
Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: new mutations and the paradox between genotype and phenotype.
Up to now eight patients with alpha-NAGA deficiency have been described. This includes the newly identified patient reported here who died unexpectedly aged 1 1/2 years of hypoxia during convulsions; necropsy was not performed. Three patients have been genotyped previously and here we report the mutations in the other five patients, including two new mutations (S160C and E193X). The newly identified patient is consanguineous with the first patients reported with alpha-NAGA deficiency and neuroaxonal dystrophy and they all had the alpha-NAGA genotype E325K/E325K. Clinical heterogeneity among patients with alpha-NAGA deficiency is extreme. Two affected sibs, homozygotes for E325K, are severely affected and have the signs and symptoms of infantile neuroaxonal dystrophy, but prominent vacuolisation is lacking. The mildly affected patients (two families, three patients) at the opposite end of the clinical spectrum have clear vacuolisation and angiokeratoma but no overt neurological manifestations. Two of them are homozygous for the stop mutation E193X, leading to complete loss of alpha-NAGA protein. These observations are difficult to reconcile with a simple genotype-phenotype correlation and we suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the eight patients with alpha-NAGA deficiency. At the metabolic level, the patients with alpha-NAGA deficiency are similar. The major abnormal urinary oligosaccharides are sialylglycopeptides of the O linked type. Our enzymatic studies indicated that these compounds are not the primary lysosomal storage products.
Animals↗
Galactose stabilizes various missense mutants of alpha-galactosidase in Fabry disease.
The effect of galactose on alpha-galactosidase missense mutants causing Fabry disease was investigated in the COS-1 cell expression system and lymphoblasts. Three mutant enzymes, A156V, L166V and Q279E, showed increases in activity and amount in COS-1 cells cultured with galactose. Another mutant without catalytic activity, C142Y, did not show any changes. In lymphoblasts cultured with galactose, the enzyme activity increased significantly in four classical Fabry patients with the respective mutations, A156V, L166V, G260A and G373S, and in three atypical Fabry patients with the respective mutations, Q279E, R301Q and M296I. Such an increase was not observed in the other four classical Fabry patients, with C142Y, E66Q/R112C, G328R and N320K, respectively. This suggests that many missense mutations in the alpha-galactosidase gene causing Fabry disease allow the expression of catalytically active mutant enzymes regardless of the clinical phenotype, which are rapidly degraded under physiological conditions and stabilized by galactose.
Amino Acid Sequence↗