GAPDH is conformationally and functionally altered in association with oxidative stress in mouse models of amyotrophic lateral sclerosis

Abstract

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.

Figure 1.. Detection of Changes in Protein Folding in Skeletal Muscle Cytosolic Proteins from G93A Mice.

The cytosolic proteins isolated from skeletal muscle of 130-day-old G93A mice were photolabeled with BisANS as described in the Methods section. Equal amounts of proteins (20 μg) of BisANS-labeled proteins were subject to SDS-PAGE and visualized the labeled proteins under UV light followed by staining with coomassie blue. Fluorescent (upper panels) and coomassie (lower panels) images are shown for G93A (left), WT SOD1 (right) and their respective NTg littermates. Regions sensitive to conformational alteration in G93A are indicated by arrows.

Figure 2.. 2D Gel Separation of BisANS Labeled Skeletal Muscle Cytosolic Proteins from G93A Mice.

The cytosolic proteins isolated from skeletal muscle of 130-day-old G93A mice were separated by 2D gel electrophoresis (150 μg), and visualized under UV light (upper panels) followed by overnight staining of the proteins with Sypro Ruby (lower panels). Spots (circled) from two regions of interest were obtained and identified by mass spectrometry.

Figure 3.. Creatine Kinase and GAPDH Activity in Skeletal Muscle from G93A Mice.

Graph A) the activities of CK (upper panel) and GAPDH (lower panel) were measured in skeletal muscle cytosol from G93A, TgSOD1, and their respective NTg littermates as described in Methods. Values are expressed as the average percent activity of NTg and represent the mean of 3 animals per group ± standard deviation *= p<0.05. Graph B) Western blots for CK and GAPDH are shown. Graph C) Western blot for CK (upper panel) and GAPDH (lower panel) in the detergent-insoluble P3 fraction of skeletal muscle lysates.

Figure 4.. Effect of Oxidative Stress In Vitro on the Activity and Conformation of GAPDH and CK.

Graph A) purified rabbit muscle CK and GAPDH (1 mg/ml) were oxidized separately with various concentrations of iron (0, 0.2, 0.4, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 20, and 40 μM) and ascorbate (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 5 and 10 mM) for 1 hr at 37°C in the dark followed by labeling with BisANS (100 μM). CK and GAPDH (5 μg, each) were mixed and separated by SDS-PAGE and the fluorescence of BisANS was visualized as described in the Methods. Shown are the CK and GAPDH BisANS fluorescence (upper panel) and Coomassie images from the gel (lower panel). Graph B) the ratio of BisANS intensity: Coomassie intensity for CK (closed circles) and GAPDH (open circles) was quantitated by densitometry and expressed as percent intensity of control (no oxidation). Graph C) enzyme activity of iron and ascorbate oxidized CK (closed circles) and GAPDH (open circles) was measured and expressed as percent activity of control (no stress). The data represent the mean of 3 experiments, +/− standard deviation.

Figure 5.. 2D Gel Separation of 6-IAF Labeled Skeletal Muscle Cytosolic Proteins from G93A Mice.

The cytosolic proteins isolated from skeletal muscle of H46R/H48Q were labeled with 6IAF as described in Methods. 6-IAF labeled skeletal muscle cytosolic proteins (150 μg) were separated by 2D gel electrophoresis, and the region containing CK and GAPDH is shown. Three separate animals were analyzed per group, and shown are representative gels from each group.

Figure 6.. Separation of BisANS Labeled Skeletal Muscle Cytosolic Proteins from H46R/H48Q Mice.

Graph A) The cytosolic proteins isolated from skeletal muscle of H46R/H48Q were photolabeled with BisANS as described in Methods. Equal amounts of BisANS-labeled proteins (20 μg) were subject to SDS-PAGE, visualized under UV and stained with coomassie blue. Graph B) BisANS labeled skeletal muscle cytosolic proteins (150 μg) were separated by 2D gel electrophoresis, and the region containing CK and GAPDH is shown. Graph C) CK (upper panel) and GAPDH (lower panel) activity are shown, and the values are expressed as the average percent activity of WT. The data represent the mean of 3 animals per group +/− standard deviation, and the values that are significantly different from WT are shown (*= p<0.05). D) Western blots for CK and GAPDH are shown.

Figure 7.. Hypothetical Reaction Scheme of BisANS Reaction with Nucleophilic Amino Acids.

A) Nucleophilic amino acids such as lysine, serine, threonine, and tyrosine undergo a nucleophilic attack on the resonance structure of the aniline ring of BisANS, forming a BisANS cross-linked amino acid. B) MS/MS analysis of peptide AENGKLVINGKPIT-IFQER from GAPDH. All of the “y” and “b” ions are assigned in the spectrum. C) Diagram showing the “y” and “b” ion map superimposed on the peptide sequence. The BisANS lysine is labeled in the sequence.

Figure 8.. X-Ray Crystal Structure of Human GAPDH with BisANS Incorporation Sites Modeled in NTg, G93A, and H46R/H48Q Mice.

BisANS cross-linked sites found by LC-MS/MS in mouse GAPDH are indicated in the stereoview of the crystal structure of tetrameric human GAPDH (pdb code 1u8f) described by Jenkins and Tanner followed by the corresponding human sites in parentheses. BisANS sites incorporated to GAPDH are shown only on one of the four subunits corresponding to the mouse amino acid number A) BisANS sites found in the NTg mouse GAPDH by LC/MS/MS are indicated by their mouse amino acid number. Amino acids involved in enzyme activity are indicated by black dots, and sites lost by both mutants are indicated by green, lost only in G93A by red, and only in H46R/H48Q in orange. B) Sites gained in G93A are indicated by red spheres, and those gained by both G93A and H46R/H48Q are indicated in cyan.

Figure 8.. X-Ray Crystal Structure of Human GAPDH with BisANS Incorporation Sites Modeled in NTg, G93A, and H46R/H48Q Mice.

BisANS cross-linked sites found by LC-MS/MS in mouse GAPDH are indicated in the stereoview of the crystal structure of tetrameric human GAPDH (pdb code 1u8f) described by Jenkins and Tanner followed by the corresponding human sites in parentheses. BisANS sites incorporated to GAPDH are shown only on one of the four subunits corresponding to the mouse amino acid number A) BisANS sites found in the NTg mouse GAPDH by LC/MS/MS are indicated by their mouse amino acid number. Amino acids involved in enzyme activity are indicated by black dots, and sites lost by both mutants are indicated by green, lost only in G93A by red, and only in H46R/H48Q in orange. B) Sites gained in G93A are indicated by red spheres, and those gained by both G93A and H46R/H48Q are indicated in cyan.

Figure 9.. Summary of Functional and Conformational Changes Observed in GAPDH in Response to Oxidative Stress.

Tetrameric human GAPDH (pdb code 1u8f) is shown with BisANS sites incorporated to GAPDH shown only on one of the four subunits. The changes in BisANS incorporation sites that correlate with the level of oxidative stress and GAPDH activity in the NTg (left), H46R/H48Q (middle), G93A (right) models are shown. The amino acids involved in enzyme activity are indicated in black. All NTg sites are indicated by green dots, and sites gained with increased oxidative stress are indicated by red dots. The sites corresponding to doubly labeled peptides are circled.