Takahiro Tsukimura

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.

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.

Use of a Modified α-N-Acetylgalactosaminidase in the Development of Enzyme Replacement Therapy for Fabry Disease

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 NAGAs 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.

Molecular interaction of imino sugars with human α-galactosidase: Insight into the mechanism of complex formation and pharmacological chaperone action in 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.

Human α-l-iduronidase uses its own N-glycan as a substrate-binding and catalytic module

N-glycosylation is a major posttranslational modification that endows proteins with various functions. It is established that N-glycans are essential for the correct folding and stability of some enzymes; however, the actual effects of N-glycans on their activities are poorly understood. Here, we show that human α-l-iduronidase (hIDUA), of which a dysfunction causes accumulation of dermatan/heparan sulfate leading to mucopolysaccharidosis type I, uses its own N-glycan as a substrate binding and catalytic module. Structural analysis revealed that the mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. A deglycosylation study showed that enzyme activity was highly correlated with the N-glycan attached to N372. The kinetics of native and deglycosylated hIDUA suggested that the N-glycan is also involved in catalytic processes. Our study demonstrates a previously unrecognized function of N-glycans.

High-throughput screening identified disease-causing mutants and functional variants of α-galactosidase A gene in Japanese male hemodialysis patients

Fabry disease is a genetic disorder caused by deficient activity of lysosomal enzyme α-galactosidase A (GLA) and end-stage renal disease (ESRD) will be present after accumulation of glycosphingolipids within the kidney. Undiagnosed atypical variants of Fabry disease, which are limited to renal involvement, were found in several ESRD patient populations. On the other hand, unexpectedly high frequencies of male subjects having the c.196G>C nucleotide change (p.E66Q) showing low α-GLA activity have been reported on Japanese and Korean screening for Fabry disease. However, several evidences indicate the c.196G>C is not a pathogenic mutation but is a functional polymorphism. In the present study, high-throughput screening of serum GLA could successfully indentify two Fabry disease patients in a cohort consisted of 1080 male hemodialysis patients. Moreover, our serum assay was able to distinguish two patients with disease-causing genetic mutations (p.G195V and p.M296I) from eight functional variants that showed relatively decreased enzyme activity (p.E66Q). In conclusion, high-throughput serum enzyme assay distinctly identified disease-causing mutants and functional variants of GLA gene in Japanese male hemodialysis patients. In addition, our results underscore the high prevalence of not only undiagnosed Fabry patients but functional variants of p.E66Q among the ESRD population.

Mutant α-galactosidase A with M296I does not cause elevation of the plasma globotriaosylsphingosine level

Recently, plasma globotriaosylsphingosine (lyso-Gb3) has attracted attention as a biomarker of Fabry disease. However, we found a subset of Fabry disease patients who did not show any increase in the plasma lyso-Gb3 concentration, although other patients exhibited apparent enhancement of it. This subset predominantly exhibited the clinical phenotype of later-onset Fabry disease, and gene analysis revealed that the patients harbored the M296I mutation common to Japanese Fabry patients. This amino acid substitution is predicted to cause a small conformational change on the surface of the α-galactosidase A molecule, resulting in residual enzyme activity. Plasma lyso-Gb3 is a good biomarker of Fabry disease but care should be taken when it is used for a definitive diagnosis.

Lyso-GM2 Ganglioside: A Possible Biomarker of Tay-Sachs Disease and Sandhoff Disease

To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.