Crystal structure of rice alpha-galactosidase complexed with D-galactose.
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alpha-Galactosidases catalyze the hydrolysis of alpha-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice alpha-galactosidase has been determined at 1.5A resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of alpha-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (beta/alpha)8-barrel structure, and the C-terminal domain was made up of eight beta-strands containing a Greek key motif. The structure was solved as a complex with d-galactose, providing a mode of substrate binding in detail. The d-galactose molecule was found bound in the active site pocket on the C-terminal side of the central beta-barrel of the catalytic domain. The d-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice alpha-galactosidase with chicken alpha-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes. (+info)
Early detection of Fabry cardiomyopathy by tissue Doppler imaging.
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BACKGROUND: Fabry cardiomyopathy is diagnosed by detection of left ventricular hypertrophy (LVH) in patients with alpha-Galactosidase A deficiency. Conventional noninvasive tools are unable to provide a preclinical diagnosis allowing prompt institution of enzymatic therapy. METHODS AND RESULTS: We studied three groups of patients: 10 patients with causal mutations for Fabry disease and LVH, 10 mutation-positive patients without LV, and 10 healthy relatives without causal mutations and no LVH. All patients with LVH and 6 patients with Fabry disease without LVH with complex repetitive ventricular arrhythmias underwent biventricular endomyocardial biopsy to assess cardiac involvement. In all patients 2-dimensional echocardiography with tissue Doppler analysis in the pulsed Doppler mode was performed: systolic (Sa), early diastolic (Ea), and late diastolic (Aa) velocities were measured, and the Ea/Aa ratio and the dimensionless parameter E/Ea were computed at both corners of the mitral annulus. Histology and electron microscopy studies showed glycosphingolipid deposits in all cases. All mutation-positive patients had significant reduction of Sa, Ea, and Aa velocities at both corners of the mitral annulus compared with normal control subjects. Ea/Aa ratio was significantly lower and E/Ea ratio significantly higher in mutation-positive patients than in control subjects. Patients with LVH showed significantly lower contraction and relaxation tissue Doppler velocities, lower Ea/Aa ratio, and higher E/Ea ratio in comparison with mutation-positive patients with no LVH. CONCLUSIONS: Fabry cardiomyopathy is characterized by reduced myocardial contraction and relaxation tissue Doppler velocities, detectable even before development of LVH. Tissue Doppler imaging can provide a preclinical diagnosis of Fabry cardiomyopathy, allowing early institution of enzyme replacement therapy. (+info)
Fabry disease: novel alpha-galactosidase A 3'-terminal mutations result in multiple transcripts due to aberrant 3'-end formation.
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Mutations in the gene that encodes the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-GalA), cause Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism. Human alpha-GalA is one of the rare mammalian genes that has its polyadenylation signal in the coding sequence and lacks a 3' untranslated region (UTR). We identified two novel frameshift mutations, 1277delAA (del2) and 1284delACTT (del4), in unrelated men with classical Fabry disease. Both mutations occurred in the 3' terminus of the coding region and obliterated the termination codon, and del2 also altered the polyadenylation signal. To characterize these mutations, 3' rapid amplification of cDNA ends (RACE) and polymerase chain reactions (PCR) were performed, and the amplicons were subcloned and sequenced. Both mutations generated multiple transcripts with various lengths of 3' terminal sequences, some elongating approximately 1 kb. Mutant transcripts were classified as follows: type I transcripts had terminal in-frame thymidines that created termination codons when polyadenylated, type II had downstream termination codons within the elongated alpha-GalA sequence, and type III, the most abundant, lacked termination codons at their 3' ends. To determine if the type III transcripts were degraded by the recently described cytosolic messenger RNA degradation pathway for messages lacking termination codons, northern blot analysis was performed. However, the finding of similar levels of nuclear and cytoplasmic alpha-GalA mRNA in normal and patient lymphoblasts suggested that mRNA degradation did not result from either mutation. Expression of representative transcript types revealed differences in intracellular localization and/or protein stability and catalytic activity, with most mutant proteins being nonfunctional. Characterization of these 3' mutations identified a novel molecular mechanism causing classical Fabry disease. (+info)
IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia.
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Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella. Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed. To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20. We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at approximately 17 S, similar to IFT proteins extracted from mouse testis. Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina. We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex. The internal architecture of the IFT complex was investigated using the yeast two-hybrid system. IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B. Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types. Furthermore, IFT20 appears to bridge kinesin II with the IFT complex. (+info)
Characterization of genes involved in the metabolism of alpha-galactosides by Lactococcus raffinolactis.
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Lactococcus raffinolactis, unlike most lactococci, is able to ferment alpha-galactosides, such as melibiose and raffinose. More than 12 kb of chromosomal DNA from L. raffinolactis ATCC 43920 was sequenced, including the alpha-galactosidase gene and genes involved in the Leloir pathway of galactose metabolism. These genes are organized into an operon containing aga (alpha-galactosidase), galK (galactokinase), and galT (galactose 1-phosphate uridylyltransferase). Northern blotting experiments revealed that this operon was induced by galactosides, such as lactose, melibiose, raffinose, and, to a lesser extent, galactose. Similarly, alpha-galactosidase activity was higher in lactose-, melibiose-, and raffinose-grown cells than in galactose-grown cells. No alpha-galactosidase activity was detected in glucose-grown cells. The expression of the aga-galKT operon was modulated by a regulator encoded by the upstream gene galR. The product of galR belongs to the LacI/GalR family of transcriptional regulators. In L. lactis, L. raffinolactis GalR acted as a repressor of aga and lowered the enzyme activity by more than 20-fold. We suggest that the expression of the aga operon in lactococci is negatively controlled by GalR and induced by a metabolite derived from the metabolism of galactosides. (+info)
The blood group P1 synthase gene is identical to the Gb3/CD77 synthase gene. A clue to the solution of the P1/P2/p puzzle.
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Blood group P1/P2 is a glycolipid antigen system for which the genetic mechanism has not yet been clarified. We analyzed the potential of the cloned Gb3/CD77 synthase to synthesize P1 antigen, because Gb3/CD77 and P1 share a common structure, Galalpha1,4Galbeta1,4Glc (NAc)-. L cell transfectants with Gb3/CD77 synthase cDNA expressed marginal levels of P1 on the cell surface but contained high levels of P1 in the cytoplasm. P2-type erythrocytes, which were serotyped as P2, also contained definite P1 antigen inside cells, although the amounts were lower than those of P1 cells. Only p erythrocytes lacked P1 antigen corresponding with function-losing mutations in the Gb3/CD77 synthase gene. Synthesis of P1 antigen from paragloboside in vitro was demonstrated using membrane fraction of the transfectants and a fusion enzyme with protein A. These results strongly suggested that P1 synthase is identical to Gb3/CD77 synthase and appear to propose a clue for the solution of the long-pending P1/P2/p puzzle. The P1/P2 difference might result from the difference in P1 quantity based on either different enzyme activity or the presence/absence of other enzyme modulators. Because P2 erythrocytes showed lower levels of Gb3/CD77 synthase mRNA than P1, 5'-upstream promoter regions were analyzed, resulting in the identification of two P2-specific homozygous mutations. Differences in the transcriptional regulation in erythrocytes might be a major factor determining P1/P2. (+info)
Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum.
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Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses. (+info)
Improvement of cardiac function during enzyme replacement therapy in patients with Fabry disease: a prospective strain rate imaging study.
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BACKGROUND: Enzyme replacement therapy (ERT) has been shown to enhance microvascular endothelial globotriaosylceramide clearance in the hearts of patients with Fabry disease. Whether these results can be translated into an improvement of myocardial function has yet to be demonstrated. METHODS AND RESULTS: Sixteen patients with Fabry disease who were treated in an open-label study with 1.0 mg/kg body weight of recombinant alpha-Gal A (agalsidase beta, Fabrazyme) were followed up for 12 months. Myocardial function was quantified by ultrasonic strain rate imaging to assess radial and longitudinal myocardial deformation. End-diastolic thickness of the left ventricular posterior wall and myocardial mass (assessed by magnetic resonance imaging, n=10) was measured at baseline and after 12 months of ERT. Data were compared with 16 age-matched healthy controls. At baseline, both peak systolic strain rate and systolic strain were significantly reduced in the radial and longitudinal direction in patients compared with controls. Peak systolic strain rate increased significantly in the posterior wall (radial function) after one year of treatment (baseline, 2.8+/-0.2 s(-1); 12 months, 3.7+/-0.3 s(-1); P<0.05). In addition, end-systolic strain of the posterior wall increased significantly (baseline, 34+/-3%; 12 months, 45+/-4%; P<0.05). This enhancement in radial function was accompanied by an improvement in longitudinal function. End-diastolic thickness of the posterior wall decreased significantly after 12 months of treatment (baseline, 13.8+/-0.6 mm; 12 months, 11.8+/-0.6 mm; P<0.05). In parallel, myocardial mass decreased significantly from 201+/-18 to 180+/-21 g (P<0.05). CONCLUSIONS: These results suggest that ERT can decrease left ventricular hypertrophy and improve regional myocardial function. (+info)