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. Mar-Apr 2013;7(2):175-84.
doi: 10.4161/pri.22992. Epub 2012 Dec 3.

Amyloidogenic Peptides of Yeast Cell Wall Glucantransferase Bgl2p as a Model for the Investigation of Its pH-dependent Fibril Formation

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Amyloidogenic Peptides of Yeast Cell Wall Glucantransferase Bgl2p as a Model for the Investigation of Its pH-dependent Fibril Formation

Evgeny E Bezsonov et al. Prion. .
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Abstract

The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence. Three PADs, FTIFVGV, SWNVLVA and NAFS, were selected on the basis of combination of computational algorithms. Peptides AEGFTIFVGV, VDSWNVLVAG and VMANAFSYWQ, containing these PADs, were synthesized. It was demonstrated that these peptides had an ability to fibrillate at pH values from 3.2 to 5.0. The PAD-containing peptides, except for VDSWNVLVAG, could fibrillate also at pH values from pH 5.0 to 7.6. We supposed that the ability of Bgl2p to form fibrils most likely depended on the coordination of fibrillation activity of the PAD-containing areas and Bgl2p could fibrillate at mild acid and neutral pH values and lose the ability to fibrillate with the increasing of pH values. It was demonstrated that Bgl2p was able to fibrillate at pH value 5.0, to form fibrils of various morphology at neutral pH values and lost the fibrillation ability at pH value 7.6. The results obtained allowed us to suggest a new simple approach for the isolation of Bgl2p from Saccharomyces cerevisiae cell wall.

Keywords: Bgl2p; amyloid; glucantransferase; thioflavin T; yeast cell wall.

Figures

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Figure 1. Potential amyloidogenic determinants in Saccharomyces cerevisiae cell wall glucantransferase Bgl2p (UniProtKB/TrEMBL entry number P15703). The amino acids predicted by computational algorithms to be a part of potential amyloidogenic determinants are marked with (*) opposite the name of the corresponding algorithm. The confluences of positive amyloidogenic determinant prediction results are surrounded by frames. Bgl2p N-terminal cell wall transport signal (in gray letters); peptide sequences, which were synthesized and investigated, are saturated gray. Abbreviations: aa, amino acid number; seq, amino acid sequence.
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Figure 2. Fluorescence microscopy of the peptides at pH 5.0. (A) The peptide (aa 166-175 “B”). (B) The peptide (aa 166-175 “NB”). Staining was done with 7.5 μM ThT.
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Figure 3. Fibrillation kinetics of Bgl2p at different pH values measured by thioflavin T fluorescence at 480 nm at pH 5.0 (solid line) and pH 7.6 (dashed line). The fibrillation kinetics was measured at 35°C and using 600 rpm of shaking for 240 sec during each cycle of 400 sec. The excitation wavelength was 450 nm. The plotted data curves are an average of the three individually measured fibrillation kinetics.
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Figure 4. Bgl2p fibril morphology. (A–E) Immunofluorescent microscopy using anti-Bgl2p antibody. Bgl2p was isolated from the CW, which was either not treated (A–C) or treated (D and E) with trypsin before Bgl2p extraction from Saccharomyces cerevisiae cell wall. (F– J) Transmission electron microscopy. Bgl2p was isolated from trypsin-treated Saccharomyces cerevisiae cell wall and then it was incubated in absence (F–H) or in presence (I and J) of laminarin. Arrows indicate the “aggregation centers.”
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Figure 5. Bgl2pGM fibril morphology. Immunofluorescent microscopy of Bgl2pGM fibrils with anti-Bgl2p antibody. Ultrafiltered growth medium of Saccharomyces cerevisiae ssu21/mcd4 mutant contains components which pass through the membrane with a cut-off limit of 100 kDa. Images of these components were collected without Congo red (A) and in presence of 8 µM Congo red (B–D). The growth medium containing components which do not pass through the membrane with a cut-off limit of 10 kDa (E).
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Figure 6. Analysis of Bgl2p which was extracted from Saccharomyces cerevisiae cell wall in 100 mM TRIS solution, pH 9.2. (A) SDS-PAGE,Coomassie staining. (B) Western blot analysis. Bgl2p was detected with antibodies. 1. Protein marker. 2. Bgl2p preparation.

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