Role of aspartate 42 and histidine 79 in the aiPLA2 activity and oligomeric status of Prdx6 at low pH

Abstract

Peroxiredoxin 6 (Prdx6), a unique non-seleno peroxidase, is a bifunctional protein with GSH peroxidase at pH 7.4 and calcium independent phospholipase A₂ (aiPLA₂) activities at pH 4.0. Changes in pH brings about alteration in the conformational and thermodynamic stability of Prdx6. For instance, under acidic condition (pH 4.0), Prdx6 forms higher oligomers with concommittant gain in aiPLA₂ activity that is resistant to thermal denaturation. However, there has been no molecular level understanding of how low pH induces formation of oligomers. In the present study, site directed mutagenesis of two conserved amino acid residues, Asp42 and His79, was used to study the molecular basis for the influence of pH on the oligomeric state of Prdx6. We observed that mutation at Asp42 and His79 residues by Ala did not result in a significant change in its peroxidase activity at neutral pH 7.4, but its aiPLA₂ activity at low pH 4.0 decreased significantly. At this pH condition, both mutants exhibit highly conserved alpha-helix content but fluctuating tryptophan micro-environment with partly exposed hydrophobic patches that render the formation of oligomers. DLS measurements and analytical SEC revealed that Wt Prdx6 forms oligomers at low pH but not the mutant proteins suggesting the importance of these residues in pH sensing and oligomerization. These results suggest that Asp42 and His79 interact each other to induce conformational change of Prdx6 that triggers the oligomerization of Prdx6 at low pH.

Keywords: π glutathione S-transferase; 1, 2,-bis palmitoyl-sn-glycero-3-phosphocholine; Calcium independent phospholipase A2; Circular dichroism; Peroxiredoxin 6; Thioredoxin.

Fig. 1

Primary structure of 1-Cys Prdx from different species. Consensus sequences from NCBI aligned using T-coffee multiple sequence alignment of EMBL-EBI. The red and yellow shading indicates regions of fully-conserved and highly-conserved regions respectively among the species. Residue numbering follows the residues of Prdx6. Black vertical box shows the fully conserved Asp residue. Red box represents the highly conserved His residue throughout the species.

Fig. 2

(A) Determination of Peroxidase activity of Prdx6 and its mutant, D42A and H79A using H₂O₂ decay assay as the extent of consumption of H₂O₂ upon reacting with Prdx6 is monitored by the decrease in absorbance at 240 nm (ε₂₄₀ for H₂O₂ being 43.6 M⁻¹ cm⁻¹). (B) The enzymatic activity estimated for Prdx6 and its mutant is expressed as nmol of reduced H₂O₂/min. mg of Prdx6. Estimation of calcium independent Phospholipase activity (aiPLA₂) of Prdx6 and its mutant as fluorescence emission plot (against time) (C) of EnzChek® Phospholipase A₂ substrate incorporated in liposomes (prepared by mixing of 30 μl 10 mM Dioleoylphosphatidylcholine, 30 μl 10 mM Dioleoylphosphatidylglycerol, and 30 μl 1 mM PLA₂ substrate) with addition of Prdx6 at temperature, 25 °C and (D) its fluorescence ratio (515/575).

Fig. 3

Dynamic light scattering (DLS) measurement as the size distribution by volume of Prdx6 and its mutant D42A and H79A at (A) pH 7.4 and (B) pH 4.0. Measurement of elution volume of Prdx6 and its mutant as absorbance at 280 nm through Size Exclusion Chromatography at pH 7.4 (C) and 4.0 (D).

Fig. 4

Thermal-induced denaturation of Prdx6 and its mutant D42A and H79A as recorded following changes in [θ]₂₂₀ from 20 °C to 80 °C at a rate of 1 °C/min using Circular Dichroism spectroscopy at pH 7.4 (A) and 4.0 (B).

Fig. 5

Conformational studies of Prdx6 and its mutant, D42A and H79A. Far-UV CD measurements at pH 7.4 (A) and pH .4.0 (B). Tryptophan fluorescence at pH 7.4 (C) and pH 4.0 (D); and ANS measurements at pH 7.4 (E) and pH 4.0 (F) of Prdx6 and its mutant were measured at standard buffer Na-Acetate buffer, pH 4.0 and 50 mM Tris–HCl, 100 mM NaCl, pH 7.4 at 25 °C. All spectra are mean of three independent experiments.

Fig. 6

Illustration of structural forms of Asp42 and His79 in different Peroxiredoxin family. Intramolecular distance between Oxygen atom of Asp42 and Nitrogen atom of His 79 in different Peroxiredoxin family. (A) Prdx6 (represented from PDB ID: 1PRDX) (B) Prdx1 (represented from PDB ID: 2Z9S (C) Prdx2 (represented from PDB ID: 7KJ0 (D) Overlapped view of Prdx6, Prdx1 and Prdx2. Both the residues of Asp42 and His79 of Prdx6 and corresponding residues of Prdx1 and Prdx2 are aligned and have similar conformation.