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. 2013 Sep 16;8(9):e74336.
doi: 10.1371/journal.pone.0074336. eCollection 2013.

Prolonged glycation of hen egg white lysozyme generates non amyloidal structures

Sudeshna Ghosh  1 , Nitin Kumar Pandey, Atanu Singha Roy, Debi Ranjan Tripathy, Amit Kumar Dinda, Swagata Dasgupta
Affiliations

Prolonged glycation of hen egg white lysozyme generates non amyloidal structures

Sudeshna Ghosh et al. PLoS One. .

Abstract

Glycation causes severe damage to protein structure that could lead to amyloid formation in special cases. Here in this report, we have shown for the first time that hen egg white lysozyme (HEWL) does not undergo amyloid formation even after prolonged glycation in the presence of D-glucose, D-fructose and D-ribose. Cross-linked oligomers were formed in all the cases and ribose was found to be the most potent among the three sugars. Ribose mediated oligomers, however, exhibit Thioflavin T binding properties although microscopic images clearly show amorphous and globular morphology of the aggregates. Our study demonstrates that the structural damage of hen egg white lysozyme due to glycation generates unstructured aggregates.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Determination of Trp fluorescence on glycation of HEWL in the presence of different sugars using fluorescence spectroscopy.
(a) Histogram represents Trp fluorescence intensity of different HEWL solutions incubated in the presence of glucose, fructose and ribose respectively over a period of 120 days. (b) Representative Trp fluorescence spectra of different HEWL solutions incubated in the presence of glucose, fructose and ribose respectively obtained after an incubation of 31 days. Control represents native HEWL incubated in the absence of sugars at pH 7.4 at 37 °C keeping other conditions similar as that of sets in each case.
Figure 2
Figure 2. Characterization of different AGE products formed during glycation of HEWL in the presence of different sugars using fluorescence spectroscopy.
Histograms represent fluorescence intensity of different HEWL solutions incubated in the presence of glucose, fructose and ribose respectively over a period of 120 days. Formation of different AGE products such as (a) other AGE products (λex=350 nm), (b) pentosidine (λex=335 nm) and (c) malondialdehyde (MDA) (λex=370 nm). [HEWL]=5 µM in each case. Control represents native HEWL incubated in the absence of sugars at pH 7.4 at 37 °C keeping other conditions similar as that of sets in each case.
Figure 3
Figure 3. Synchronous fluorescence characteristics of HEWL during glycation in the presence of different sugars.
(a) Synchronous fluorescence spectra of control and HEWL solutions treated in the presence of glucose, fructose and ribose respectively after 31 days of incubation at 37 °C at pH 7.4; (b) Second derivative plot of synchronous fluorescence spectra of HEWL solutions incubated in the presence of glucose, fructose and ribose respectively obtained after 31 days of incubation at 37 °C at pH 7.4. Protein concentration=10 µM and Δλ=40 nm in each case.
Figure 4
Figure 4. Detection of oligomerization of HEWL during glycation in the presence of glucose, fructose and ribose.
Representative SDS polyacrylamide gel electrophoresis of different HEWL solutions obtained after incubation at pH 7.4 at 37 °C in the presence of different sugars at definite intervals of time (1 day, 20 days and 31 days respectively). (a) In each case lane 1: molwt marker; lane 2-4: HEWL-glucose; lane: 5-7: HEWL-fructose respectively; (b) In each case lane 1: molwt marker; lane 2-4: HEWL-ribose; lane: 5: Control (native HEWL incubated at pH 7.4 at 37 °C in the absence of sugars) respectively.
Figure 5
Figure 5. Densitometric analysis of SDS-PAGE.
Histograms represent relative mean band intensity of different oligomeric species (dimer, trimer and tetramer) with respect to their corresponding monomer at definite time intervals (a) 1 day, (b) 20 days and (c) 31 days respectively.
Figure 6
Figure 6. Variation of secondary structural components during glycation of HEWL in the presence of different sugars.
Representative far UV-CD spectra of (a) HEWL-glucose, (b) HEWL-fructose and (c) HEWL-ribose solutions respectively obtained after incubation at pH 7.4 at 37 °C at different time intervals. [HEWL]=20 µM in each case.
Figure 7
Figure 7. Estimation of β-sheet content of HEWL solutions incubated in the presence of different sugars.
Percentage β-sheet content of different HEWL solutions (HEWL-glucose, HEWL-fructose and HEWL-ribose) obtained after incubation at pH 7.4 at 37 °C estimated using online server DICHROWEB at different time intervals.
Figure 8
Figure 8. Tertiary structural alterations of HEWL solutions during glycation in the presence of three different sugars.
Representative near UV-CD spectra of (a) HEWL-glucose, (b) HEWL-fructose and (c) HEWL-ribose solutions respectively obtained after incubation at pH 7.4 at 37 °C at different time intervals.
Figure 9
Figure 9. Identification of attachment of sugar moieties to HEWL: Fuchsin based SDS PAGE.
Representative SDS polyacrylamide gel electrophoresis of different HEWL solutions obtained after incubation at pH 7.4 at 37 °C for 31 days in the presence of different sugars using Fuchsin staining. lane 1: Horseradish peroxidase; lane 2 and 5: HEWL-glucose; lane 3 and 6: HEWL-fructose; lane 4 and 7: HEWL-ribose.
Figure 10
Figure 10. Determination of the mass of glycated HEWL.
MALDI TOF spectra of different HEWL solutions obtained after an incubation of 31 days at pH 7.4 at 37 °C (a) Native HEWL (14345.30 Da) (b) HEWL-glucose (15401.22 Da) (c) HEWL-fructose (15955.81 Da) (d) HEWL-ribose (16233.48 Da).
Figure 11
Figure 11. Variation in the ThT intensity during glycation of HEWL in the presence of three different sugars.
Histogram represents ThT fluorescence of different HEWL solutions after incubation at pH 7.4 at 37 °C at different time intervals in the presence of different sugars such as glucose, fructose and ribose respectively.
Figure 12
Figure 12. Identification of the nature of the aggregates formed during glycation of HEWL.
FESEM (a-d) and TEM images (e-h) of different HEWL solutions obtained after an incubation at pH 7.4 at 37 °C at different time intervals in the presence of different sugars. For FESEM images scale bars represent 2 µm for HEWL-glucose and HEWL-fructose; 20 µm for Control and HEWL-ribose respectively. For TEM images scale bars represent 200 nm.
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Grants and funding

SD is grateful to the Department of Science and Technology, Government of India for financial assistance for the chemicals and reagents. SG, ASR, DRT and AKD thank Council of Scientific and Industrial Research, New Delhi and NKP thank IIT Kharagpur for their senior research fellowship respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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