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. 2017 Feb 28;18(3):520.
doi: 10.3390/ijms18030520.

Ovalbumin with Glycated Carboxyl Groups Shows Membrane-Damaging Activity

Ching-Chia Tang  1 , Yi-Jun Shi  2 , Ying-Jung Chen  3 , Long-Sen Chang  4   5
Affiliations

Ovalbumin with Glycated Carboxyl Groups Shows Membrane-Damaging Activity

Ching-Chia Tang et al. Int J Mol Sci. .

Abstract

The aim of the present study was to investigate whether glycated ovalbumin (OVA) showed novel activity at the lipid-water interface. Mannosylated OVA (Man-OVA) was prepared by modification of the carboxyl groups with p-aminophenyl α-dextro (d)-mannopyranoside. An increase in the number of modified carboxyl groups increased the membrane-damaging activity of Man-OVA on cell membrane-mimicking vesicles, whereas OVA did not induce membrane permeability in the tested phospholipid vesicles. The glycation of carboxyl groups caused a notable change in the gross conformation of OVA. Moreover, owing to their spatial positions, the Trp residues in Man-OVA were more exposed, unlike those in OVA. Fluorescence quenching studies suggested that the Trp residues in Man-OVA were located on the interface binds with the lipid vesicles, and their microenvironment was abundant in positively charged residues. Although OVA and Man-OVA showed a similar binding affinity for lipid vesicles, the lipid-interacting feature of Man-OVA was distinct from that of OVA. Chemical modification studies revealed that Lys and Arg residues, but not Trp residues, played a crucial role in the membrane-damaging activity of Man-OVA. Taken together, our data suggest that glycation of carboxyl groups causes changes in the structural properties and membrane-interacting features of OVA, generating OVA with membrane-perturbing activities at the lipid-water interface.

Keywords: carboxyl group; glycation; membrane-damaging activity; ovalbumin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Membrane-perturbing activity of ovalbumin (OVA) and Man-OVA on EYPC/EYSM, EYPC/EYSM/Chol, PBPS/EYPE, and PBPS/EYPE/Chol vesicles. The experiments were performed in 10 mM Tris–HCl-0.1 M NaCl (pH 7.5). The concentrations of phospholipid vesicles were 15 μM.
Figure 2
Figure 2
Man-OVA and OVA have different Trp fluorescence spectra, thermal stability, and susceptibility for acrylamide and KI quenching. The experiments were measured using an exciting wavelength at 295 nm, and the protein concentration was 0.1 μM. (A) Intrinsic fluorescence spectra of OVA and Man-OVA; (B) Effect of temperature on Trp fluorescence of OVA and Man-OVA(600). Trp fluorescence quenching of OVA and Man-OVA(600) by acrylamide (C) and iodide (D).
Figure 3
Figure 3
The binding capability of OVA and Man-OVA(600) with phospholipid vesicles. Binding of OVA and Man-OVA(600) with phospholipid vesicles enhanced the fluorescence intensity of (A) FPE/EYPC/EYSM; (B) FPE/EYPC/EYSM/Chol; (C) FPE/PBPS/EYPE; and (D) FPE/PBPS/EYPE/Chol vesicles. The lipid concentration was 81.4 μM. The fluorescence emission intensity at 510 nm was measured.
Figure 4
Figure 4
Colorimetric dose-response curve of PDA/EYPC/EYSM, PDA/EYPC/EYSM/Chol, PDA/PBPS/EYPE, and PDA/PBPS/EYPE/Chol vesicles, titrated with Man-OVA(600) and OVA. The experiments were conducted according to the procedure described in the Materials and Methods section. The total lipid concentration of the PDA/EYPC/EYSM, PDA/EYPC/EYSM/Chol, PDA/PBPS/EYPE, and PDA/PBPS/EYPE/Chol solution was 0.5 mM.
Figure 5
Figure 5
Membrane-damaging activity of Trp-, Lys-, and Arg-modified Man-OVA(600). Man-OVA(600) and modified Man-OVA(600) induced calcein release from EYPC/EYSM, EYPC/EYSM/Chol, PBPS/EYPE, and PBPS/EYPE/Chol vesicles (* p < 0.05). The concentrations of protein and lipid vesicles were 0.2 and 15 μM, respectively.
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