α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and β-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.
- MeSH
- alfa-galaktosidasa chemie genetika izolace a purifikace metabolismus MeSH
- Basidiomycota klasifikace enzymologie genetika růst a vývoj MeSH
- Cryptococcus MeSH
- cytosol enzymologie MeSH
- DNA fungální genetika MeSH
- fungální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- geny hub genetika MeSH
- glykosidhydrolasy metabolismus MeSH
- komplementární DNA MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- laktosa metabolismus MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Fabry disease (FD) is a genetic disorder characterized by accumulation of trihexosylceramide in lysosomes of various tissues leading to multiorgan manifestations, including progressive renal disease. Previous screening studies have shown that a non-neglectable proportion of haemodialysis(HD) patients have unsuspected FD. An extensive FD screening study, the largest to date, has been conducted in HD patients in Czech Republic. We aimed to uncover previously undiagnosed FD patients, to enable them to benefit from cause-specific therapeutic intervention with enzyme replacement therapy (ERT). METHODS: Large-scale screening was executed using a convenient automated enzymatic (alpha-galactosidose A, alpha-Gal A) dried blood spot on filter paper fluorescence method. RESULTS: In total, 3370 (45.1% males, 54.9% females) out of 4058 HD patients (83%) in Czech Republic participated in this blood spot screening (BSS) study. Abnormal low fluorescence readings were obtained in 117 patients (3.5%). Subsequent determination of plasma alpha-Gal A activity identified four males and three females with deficient plasma enzyme activity. Determination of alpha-Gal A activity in peripheral blood leucocytes and confirmatory molecular analysis resulted in four newly diagnosed Fabry males and one female. Subsequent family screening identified 10 family members with genotypically proven FD. Based on these screening results, ERT could be offered to five male FD patients. CONCLUSIONS: BSS represents a promising screening tool that has proven to be convenient and effective in uncovering unrecognized FD patients among the chronic HD population in Czech Republic.
- MeSH
- alfa-galaktosidasa chemie MeSH
- dialýza ledvin metody MeSH
- dospělí MeSH
- enzymy terapeutické užití MeSH
- Fabryho nemoc diagnóza krev terapie MeSH
- financování organizované MeSH
- fluorescenční spektrometrie MeSH
- konformace proteinů MeSH
- lidé středního věku MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- rodokmen MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH
