Residual activity of α-galactosidase A in Fabry's disease

The α-galactosidase A activity from fibroblasts of five Fabry patients and five controls has been separated from α-galactosidase B through small DEAE-cellulose columns and in some experiments by treatment of the fibroblast extracts with Sepharose coupled to anti-α-galactosidase B antibodies. By these independent methods, it has been shown that there is a residual α-galactosidase A in Fabry's disease, which is immunologically similar to the α-galactosidase A from the controls. The α-galactosidase A from all of the patients and controls has the same apparent K_m value for the synthetic substrate 4-methylumbelliferyl-α-galactoside. Four out of five patients have a thermostable α-galactosidase A, while the fifth has a thermolabile enzyme like that from the controls. The amount of immunologically active α-galactosidase A seems to be decreased in the patients tested.     

Receptor-mediated endocytosis of α-galactosidase A in human podocytes in Fabry disease

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs.     

Fabry disease: correlation between structural changes in α-galactosidase, and clinical and biochemical phenotypes

Fabry disease comprises classic and variant phenotypes. The former needs early enzyme replacement therapy, and galactose infusion is effective for some variant cases. Attempts of early diagnosis before manifestations appear will begin in the near future. However, it is difficult to predict the phenotype, to determine the therapeutic approach, only from genetic information. Thus we attempted structural analysis from a novel viewpoint. We built structural models of mutant -galactosidases resulting from 161 missense mutations (147 classic and 14 variant), and evaluated the influence of each replacement on the structure by calculating the numbers of atoms affected. Among them, 11 mutants, biochemically characterized, were further investigated by color imaging of the influenced atoms. In the variant group, the number of atoms influenced by amino-acid replacement was small, especially in the main chain. In 85% of the cases, less than three atoms in the main chain are influenced. In this group, small structural changes, located apart from the active site, result in destabilization of the mutant enzymes, but galactose can stabilize them. Structural changes caused by classic Fabry mutations are generally large or are located in functionally important regions. In 82% of the cases, three atoms or more in the main chain are affected. The classic group comprises dysfunctional and unstable types, and galactose is not expected to stabilize the mutant enzymes. This study demonstrated the correlation of structural changes, and clinical and biochemical phenotypes. Structural investigation is useful for elucidating the bases of Fabry disease and clinical treatment.     

Docking and SAR studies of calystegines: Binding orientation and influence on pharmacological chaperone effects for Gaucher’s disease

We report on the identification of the required configuration and binding orientation of nor-tropane alkaloid calystegines against β-glucocerebrosidase. Calystegine B₂ is a potent competitive inhibitor of human lysosomal β-glucocerebrosidase with K_i value of 3.3 μM. A molecular docking study revealed that calystegine B₂ had a favorable van der Waals interactions (Phe128, Trp179, and Phe246) and the hydrogen bonding (Glu235, Glu340, Asp127, Trp179, Asn234, Trp381 and Asn396) was similar to that of isofagomine. All calystegine isomers bound into the same active site as calystegine B₂ and the essential hydrogen bonds formed to Asp127, Glu235 and Glu340 were maintained. However, their binding orientations were obviously different. Calystegine A₃ bound to β-glucocerebrosidase with the same orientations as calystegine B₂ (Type 1), while calystegine B₃ and B₄ had different binding orientations (Type 2). It is noteworthy that Type 1 orientated calystegines B₂ and A₃ effectively stabilized β-glucocerebrosidase, and consequently increased intracellular β-glucocerebrosidase activities in N370S fibroblasts, while Type 2 orientated calystegines B₃ and B₄ could not keep the enzyme activity. These results clearly indicate that the binding orientations of calystegines are changed by the configuration of the hydroxyl groups on the nor-tropane ring and the suitable binding orientation is a requirement for achieving a strong affinity to β-glucocerebrosidase.     

Novel α-galactosidase A mutation in patients with severe cardiac manifestations of Fabry disease

Fabry disease (FD) is a hereditary metabolic disorder caused by the partial or total inactivation of α-galactosidase A (α-gal A), a lysosomal hydrolase. This inactivation is responsible for the accumulation of undegraded glycosphingolipids in the lysosomes with subsequent cellular and microvascular dysfunction. Fabry is considered a rare disease, with an incidence of 1:40,000; however, there are good reasons to believe that it is often seen but rarely diagnosed. To date, more than 600 mutations have been identified in human GLA gene that are responsible for FD.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Sequencing and characterization of the porcine α-galactosidase A gene: towards the generation of a porcine model for Fabry disease

Fabry disease is an inherited lysosomal disorder caused by a deficiency of alpha-galactosidase A (α-gal A). The systemic accumulation of substrate, mainly globotriaosylceramide (Gb3), results in organ failure. Although Gb3 accumulation has been observed in an α-gal A-deficient mouse model, important clinical manifestations were not seen. The pursuit of effective treatment for Fabry disease through gene therapy, for example, has been hampered by the lack of a relevant large animal model to assess the efficacy and safety of novel therapies. Towards assembling the tools to generate an alternative animal model, we have sequenced and characterized the porcine ortholog of the α-gal A gene. When compared to the human α-gal A, the porcine α-gal A showed a high level of homology in the coding regions and located at chromosome Xq22. Cell lysate and supernatants from Fabry patient-derived fibroblasts transduced with a lentiviral vector (LV) carrying the porcine α-gal A cDNA (LV/porcine α-gal A), showed high levels of α-gal A activity and its enzymological stability was similar to that of human α-gal A. Uptake of secreted porcine α-gal A was observed into non-transduced cells and was partially inhibited by soluble mannose-6-phosphate. Furthermore, Gb3 accumulation was reduced in Fabry patient-derived fibroblasts transduced with the LV/porcine α-gal A. In conclusion, we elucidated and characterized the porcine α-gal A gene and enzyme. Similarity in enzymatic profile and chromosomal location between α-gal A of porcine and human origins may be of great advantage for the development of a large animal model for Fabry disease.     

The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.     

Arginine in α-defensins: differential effects on bactericidal activity correspond to geometry of membrane curvature generation and peptide-lipid phase behavior

The conserved tridisulfide array of the α-defensin family imposes a common triple-stranded β-sheet topology on peptides that may have highly diverse primary structures, resulting in differential outcomes after targeted mutagenesis. In mouse cryptdin-4 (Crp4) and rhesus myeloid α-defensin-4 (RMAD4), complete substitutions of Arg with Lys affect bactericidal peptide activity very differently. Lys-for-Arg mutagenesis attenuates Crp4, but RMAD4 activity remains mostly unchanged. Here, we show that the differential biological effect of Lys-for-Arg replacements can be understood by the distinct phase behavior of the experimental peptide-lipid system. In Crp4, small-angle x-ray scattering analyses showed that Arg-to-Lys replacements shifted the induced nanoporous phases to a different range of lipid compositions compared with the Arg-rich native peptide, consistent with the attenuation of bactericidal activity by Lys-for-Arg mutations. In contrast, such phases generated by RMAD4 were largely unchanged. The concordance between small-angle x-ray scattering measurements and biological activity provides evidence that specific types of α-defensin-induced membrane curvature-generating tendencies correspond directly to bactericidal activity via membrane destabilization.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 5.83 mM). Among metal ions and reagents tested, Ca²⁺ and K⁺ enhanced the enzymatic activity, but Mg²⁺, Mn²⁺, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag⁺, Hg²⁺ and Co²⁺ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose.     

A systematic selection of the non-aqueous phase in a bacterial two liquid phase bioreactor treating α-pinene

A systematic evaluation of the selection criteria of non-aqueous phases in two liquid phase bioreactors (TLPBs), also named two-phase partitioning bioreactors (TPPBs), was carried out using the biodegradation of α-pinene by Pseudomonas fluorescens NCIMB 11671 as a model process. A preliminary solvent screening was thus carried out among the most common non-aqueous phases reported in literature for volatile organic contaminants biodegradation in TLPBs: silicon oil, paraffin oil, hexadecane, diethyl sebacate, dibutyl-phtalate, FC 40, 1,1,1,3,5,5,5-heptamethyltrisiloxane (HMS), and 2,2,4,4,6,8,8-heptamethylnonane (HMN). FC 40, silicone oil, HMS, and HMN were first selected based on its biocompatibility, resistance to microbial attack, and α-pinene mass transport characteristics. FC 40, HMS, HMN, and silicone oil at 10% (v/v) enhanced α-pinene mass transport from the gas to the liquid phase by a factor of 3.8, 14.8, 11.4, and 8.6, respectively, compared to a single-phase aqueous system. FC 40 and HMN were finally compared for their ability to enhance α-pinene biodegradation in a mechanically agitated bioreactor. The use of FC 40 or HMN (both at 10% v/v) sustained non-steady state removal efficiencies (RE) and elimination capacities (EC) approximately 7 and 12 times higher than those achieved in the system without an organic phase, respectively. In addition, preliminary results showed that P fluorescens could uptake and mineralize α-pinene directly from the non aqueous phase.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Taxifolin prevents postprandial hyperglycemia by regulating the activity of α-amylase: Evidence from an in vivo and in silico studies

There has been a dramatic increase in the prevalence of diabetes mellitus (DM) and its associated complications globally. The postprandial stage of DM involves prompt elevation in the levels of blood glucose and α-amylase, a carbohydrate-metabolizing enzyme is mainly involved in the regulation of postprandial hyperglycemia. This study was designed to assess the ability of a well-known flavonoid, taxifolin (TFN), against postprandial hyperglycemia and its inhibitory effects on α-amylase activity through the assessment of therapeutic potentials of TFN in an alloxan-induced diabetic animal model. The binding potential TFN with an α-amylase receptor was also investigated through molecular dynamics (MD) simulation and docking of to compare the binding affinities and energies of TFN and standard drug acarbose (ACB) with target enzyme. TFN significantly improved the postprandial hyperglycemia, lipid profile, and serum levels of α-amylase, lipase, and C-reactive protein in a dose-dependent manner when compared with that of either DM-induced and ACB-treated alloxan-induced diabetic rats. Moreover, TFN also enhanced the anti-oxidant status and normal functioning of the liver in alloxan-induced diabetic rats more efficiently as compared to that of ACB-treated alloxan-induced diabetic rats. Therapeutic potentials of TFN were also verified by MD simulation and docking results, which exhibited that the binding energy and affinity of TFN to bind with receptor was significantly higher as compared to that of ACB. Hence, the results of this study signify that TFN might be a potent inhibitor of α-amylase that has the potential to regulate the postprandial hyperglycemia along with its anti-inflammatory and anti-oxidant properties during the treatment of DM.     

The role of β-carotene and vitamin A in atherogenesis: Evidences from preclinical and clinical studies

Atherosclerotic cardiovascular disease (ASCVD) is the principal contributor to myocardial infarction, the leading cause of death worldwide. Epidemiological and mechanistic studies indicate that β-carotene and its vitamin A derivatives stimulate lipid catabolism in several tissues to reduce the incidence of obesity, but their roles within ASCVD are elusive. Herein, we review the mechanisms by which β-carotene and vitamin A modulate ASCVD. First, we summarize the current knowledge linking these nutrients with epidemiological studies and lipoprotein metabolism as one of the initiating factors of ASCVD. Next, we focus on different aspects of vitamin A metabolism in immune cells such as the mechanisms of carotenoid uptake and conversion to the vitamin A metabolite, retinoic acid. Lastly, we review the effects of retinoic acid on immuno-metabolism, differentiation, and function of macrophages and T cells, the two pillars of the innate and adaptive immune response in ASCVD, respectively.This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.