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In Fabry disease, large amounts of globotriaosylceramide (Gb3) and related glycosphingolipids accumulate in organs due to a deficiency of α-galactosidase A (GLA) activity. Enzyme replacement therapy (ERT) with recombinant GLA is now available, and it has been reported that ERT is beneficial for patients with Fabry disease, especially those who start treatment at an early stage of the disease. However, it seems that the efficacy of ERT differs with each organ, and Gb3 accumulated in the kidneys shows resistance to ERT when it is started at a late stage. In this study, we examined the differences in cleavage of Gb3 isoforms, and lyso-Gb3 and its analogues in the kidneys, liver, and heart in young Fabry mice subjected to ERT. The results revealed that recurrent administration of recombinant GLA had prominent effects in terms of degradation of Gb3 and its derivatives accumulated in the organs. However, particular Gb3 isoforms, i.e., Gb3 (C20:0) and Gb3 (C24OH), accumulated in the kidneys largely escaped from degradation. Such Gb3 isoforms may gradually accumulate in the kidneys from a young age, which results in a reduction in the efficacy of ERT for Fabry disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new 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., (Copyright 2010 Elsevier Inc. All rights reserved.)

A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.

Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant alpha-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease.

Peripheral neuropathy is one of the important manifestations of Fabry disease. Enzyme replacement therapy with presently available recombinant alpha-galactosidases does not always improve the Fabry neuropathy. But the reason has not been determined yet. We established a Schwann cell line from Fabry mice, characterized it, and then examined the uptake of alpha-galactosidase by cells and its effect on the degradation of accumulated substrate. The cells exhibited a distinct Schwann cell morphology and biochemical phenotype (alpha-Galactosidase activity was deficient, and numerous cytoplasmic inclusion bodies were present in the cells). A recombinant alpha-galactosidase added to the culture medium was incorporated into the cultured Fabry Schwann cells dose dependently. But the increase in cell-associated enzyme activity was less than that in the cases of human and mouse Fabry fibroblasts. The administration of a high dose of the enzyme improved the pathological changes in cells, although a low dose of it did not. Cellular uptake of the enzyme was strongly inhibited in the presence of mannose 6-phosphate. This suggests that the enzyme is incorporated via cation-independent mannose 6-phosphate receptors in Schwann cells. The low expression of cation-independent mannose 6-phosphate receptors in Schwann cells must be one of the reasons their uptake of the present enzymes was low. The administration of a high dose of the enzyme or the development of an enzyme containing many mannose 6-phosphate residues is required to improve Fabry neuropathy.

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-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 alpha-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.

We have previously reported the production of a recombinant alpha-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant alpha-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant alpha-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.

Peripheral neuropathy is one of the important manifestations of Fabry disease. Enzyme replacement therapy with presently available recombinant α-galactosidases does not always improve the Fabry neuropathy. But the reason has not been determined yet. We established a Schwann cell line from Fabry mice, characterized it, and then examined the uptake of α-galactosidase by cells and its effect on the degradation of accumulated substrate. The cells exhibited a distinct Schwann cell morphology and biochemical phenotype (α-Galactosidase activity was deficient, and numerous cytoplasmic inclusion bodies were present in the cells). A recombinant α-galactosidase added to the culture medium was incorporated into the cultured Fabry Schwann cells dose dependently. But the increase in cell-associated enzyme activity was less than that in the cases of human and mouse Fabry fibroblasts. The administration of a high dose of the enzyme improved the pathological changes in cells, although a low dose of it did not. Cellular uptake of the enzyme was strongly inhibited in the presence of mannose 6-phosphate. This suggests that the enzyme is incorporated via cation-independent mannose 6-phosphate receptors in Schwann cells. The low expression of cation-independent mannose 6-phosphate receptors in Schwann cells must be one of the reasons their uptake of the present enzymes was low. The administration of a high dose of the enzyme or the development of an enzyme containing many mannose 6-phosphate residues is required to improve Fabry neuropathy. [ABSTRACT FROM AUTHOR]

Abstract: Background: A unique adult male patient who developed cardiomyopathy was first suspected to have cardiac Fabry disease based on the pathological findings in heart tissues obtained on biopsy, but the α-galactosidase activity in his leukocytes was normal and no mutation was detected in the coding region of the α-galactosidase gene. We identified accumulated materials in the myocardium of this patient. Methods: Pathological and biochemical analyses were performed using the autopsied heart tissues as samples. Results: Although numerous lamellar and concentric inclusion bodies were ultrastructurally found in the autopsied myocardium, the α-galactosidase activity in the heart tissues was not decreased. Lipid analysis revealed the accumulation of phospholipids including phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, but not globotriaosylcereamide or gangliosides. Conclusions: We found that a large amount of phospholipids accumulated in the myocardium of a patient with idiopathic cardiomyopathy, and electron microscopic findings of lamellar and concentric inclusion bodies in cardiomyocytes. A cardiac phospholipid storage disorder should be considered as an important candidate disease on differential diagnosis of myocardiac disorders including cardiac Fabry disease. [Copyright &y& Elsevier]

We have previously reported the production of a recombinant α-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant α-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant α-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease. [ABSTRACT FROM AUTHOR]