Hitoshi Sakuraba

Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann–Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.

Plasma globotriaosylsphingosine as a biomarker of Fabry disease

Fabry disease is an X-linked genetic disorder caused by a deficiency of alpha-galactosidase A (GLA) activity. As enzyme replacement therapy (ERT) involving recombinant GLAs has been introduced for this disease, a useful biomarker for diagnosis and monitoring of therapy has been strongly required. We measured globotriaosylsphingosine (lyso-Gb3) and globotriaosylceramide (Gb3) in plasma samples from ten hemizygous males (six classic and four variant cases) and eight heterozygous females with Fabry disease, and investigated the responses of plasma lyso-Gb3 and Gb3 in a male Fabry patient who had undergone ERT for 4years to determine whether plasma lyso-Gb3 and Gb3 could be biomarkers of Fabry disease. The results revealed that plasma lyso-Gb3 was apparently increased in male patients and was higher in cases of the classic form than those of the variant one. In Fabry females, plasma lyso-Gb3 was moderately increased in both symptomatic and asymptomatic cases, and there was a correlation between the increase in lyso-Gb3 and the decrease in GLA activity. As to plasma Gb3, the levels in the variant Fabry hemizygotes and Fabry heterozygotes could not be distinguished from those in the controls, although those in the classic Fabry hemizygotes were increased. The plasma lyso-Gb3 level in the Fabry patient who had received ERT was elevated at the baseline and fell more dramatically on ERT than that of Gb3. Plasma lyso-Gb3 could thus be a potential biomarker of Fabry disease.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice

AbstractWe compared two recombinant α-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific α-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h−1 mg protein−1, respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in α-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Endothelin-1 in the brain of patients with galactosialidosis: its abnormal increase and distribution pattern.

Endothelin‐1 is a peptidic substrate in vitro of lysosomal protective protein/cathepsin A (PPCA) with serine carboxypeptidase activity. Endothelin‐1–specific immunoreactivity has been demonstrated to be markedly increased and distributed abnormally in the neurons and glial cells within autopsied brain regions, including the cerebellum, hippocampal formation, and spinal cord, of patients affected with galactosialidosis, a human PPCA deficiency. The genetic defect of the endothelin‐1 degrading activity of PPCA is suggested to cause some of the neurological abnormalities of this disease. Ann Neurol 2000; 47:122–126

Highly Phosphomannosylated Enzyme Replacement Therapy for GM2 Gangliosidosis

Novel recombinant human lysosomal β‐hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay‐Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency.

Structural and immunocytochemical studies on α-N-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease)

Abstractα-N-Acetylgalactosaminidase (α-NAGA) deficiency (Schindler/Kanzaki disease) is a clinically and pathologically heterogeneous genetic disease with a wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease) and late onset angiokeratoma corporis diffusum (Kanzaki disease). In α-NAGA deficiency, there are discrepancies between the genotype and phenotype, and also between urinary excretion products (sialyl glycoconjugates) and a theoretical accumulated material (Tn-antigen; Gal NAcα1-O-Ser/Thr) resulting from a defect in α-NAGA. As for the former issue, previously reported genetic, biochemical and pathological data raise the question whether or not E325K mutation found in Schindler disease patients really leads to the severe phenotype of α-NAGA deficiency. The latter issue leads to the question of whether α-NAGA deficiency is associated with the basic pathogenesis of this disease. To clarify the pathogenesis of this disease, we performed structural and immunocytochemical studies. The structure of human α-NAGA deduced on homology modeling is composed of two domains, domain I, including the active site, and domain II. R329W/Q, identified in patients with Kanzaki disease have been deduced to cause drastic changes at the interface between domains I and II. The structural change caused by E325K found in patients with Schindler disease is localized on the N-terminal side of the tenth β-strand in domain II and is smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that the main lysosomal accumulated material in cultured fibroblasts from patients with Kanzaki disease is Tn-antigen. These data suggest that a prototype of α-NAGA deficiency in Kanzaki disease and factors other than the defect of α-NAGA may contribute to severe neurological disorders, and Kanzaki disease is thought to be caused by a single enzyme deficiency.

Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Tissue and plasma globotriaosylsphingosine could be a biomarker for assessing enzyme replacement therapy for Fabry disease.

Fabry disease is a genetic disease caused by a deficiency of alpha-galactosidase A (GLA), which leads to systemic accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). With the introduction and spread of enzyme replacement therapy (ERT) with recombinant GLAs for this disease, a useful biomarker for assessing the response to ERT is strongly required. We measured the tissue level of lyso-globotriaosylsphingosine (lyso-Gb3) in Fabry mice by means of high performance liquid chromatography, and compared it with the Gb3 level. The results revealed a marked increase in the lyso-Gb3 level in most tissues of Fabry mice, and which decreased after the administration of a recombinant GLA as in the case of Gb3, which is usually used as a biomarker of Fabry disease. The response was more impressive for lyso-Gb3 compared with for Gb3, especially in kidney tissues, in which a defect significantly influences the morbidity and mortality in patients with this disease. The plasma level of lyso-Gb3 also decreased after the injection of the enzyme, and it was well related to the degradation of tissue lyso-Gb3. Thus, lyso-Gb3 is expected to be a useful new biomarker for assessing the response to ERT for Fabry disease.

Structural and biochemical studies on Pompe disease and a “pseudodeficiency of acid α-glucosidase”

AbstractWe constructed structural models of the catalytic domain and the surrounding region of human wild-type acid α-glucosidase and the enzyme with amino acid substitutions by means of homology modeling, and examined whether the amino acid replacements caused structural and biochemical changes in the enzyme proteins. Missense mutations including p.R600C, p.S619R and p.R437C are predicted to cause apparent structural changes. Nonsense mutation of p.C103X terminates the translation of acid α-glucosidase halfway through its biosynthesis and is deduced not to allow formation of the active site pocket. The mutant proteins resulting from these missense and nonsense mutations found in patients with Pompe disease are predictably unstable and degraded quickly in cells. The structural change caused by p.G576S is predicted to be small, and cells from a subject homozygous for this amino acid substitution exhibited 15 and 11% of the normal enzyme activity levels for an artificial substrate and glycogen, respectively, and corresponding amounts of the enzyme protein on Western blotting. No accumulation of glycogen was found in organs including skeletal muscle in the subject, and thus the residual enzyme activity could protect cells from glycogen storage. On the other hand, p.E689K, which is known as a neutral polymorphism, little affected the three-dimensional structure of acid α-glucosidase. Structural study on a mutant acid α-glucosidase in silico combined with biochemical investigation is useful for understanding the molecular pathology of Pompe disease.

Fabry disease: Biochemical, pathological and structural studies of the α-galactosidase A with E66Q amino acid substitution

Recently, male subjects harboring the c.196G>C nucleotide change which leads to the E66Q enzyme having low α-galactosidase A (GLA) activity have been identified at an unexpectedly high frequency on Japanese and Korean screening for Fabry disease involving dry blood spots and plasma/serum samples. Individuals with the E66Q enzyme have been suspected to have the later-onset Fabry disease phenotype leading to renal and cardiac disease. However, there has been no convincing evidence for this. To determine whether c.196G>C (E66Q) is disease-causing or not, we performed biochemical, pathological and structural studies. It was predicted that the E66Q amino acid substitution causes a small conformational change on the molecular surface of GLA, which leads to instability of the enzyme protein. However, biochemical studies revealed that subjects harboring the E66Q enzyme exhibited relatively high residual enzyme activity in white blood cells, and that there was no accumulation of globotriaosylceramide in cultured fibroblasts or an increased level of plasma globotriaosylsphingosine in these subjects. An electron microscopic examination did not reveal any pathological changes specific to Fabry disease in biopsied skin tissues from a male subject with the E66Q enzyme. These results strongly suggest that the c.196G>C is not a pathogenic mutation but is a functional polymorphism.