Protein Carbonylation and Glycation in Legume Nodules

Abstract

Nitrogen fixation is an agronomically and environmentally important process catalyzed by bacterial nitrogenase within legume root nodules. These unique symbiotic organs have high metabolic rates and produce large amounts of reactive oxygen species that may modify proteins irreversibly. Here, we examined two types of oxidative posttranslational modifications of nodule proteins: carbonylation, which occurs by direct oxidation of certain amino acids or by interaction with reactive aldehydes arising from cell membrane lipid peroxides; and glycation, which results from the reaction of lysine and arginine residues with reducing sugars or their autooxidation products. We used a strategy based on the enrichment of carbonylated peptides by affinity chromatography followed by liquid chromatography-tandem mass spectrometry to identify 369 oxidized proteins in bean (Phaseolus vulgaris) nodules. Of these, 238 corresponded to plant proteins and 131 to bacterial proteins. Lipid peroxidation products induced most carbonylation sites. This study also revealed that carbonylation has major effects on two key nodule proteins. Metal-catalyzed oxidation caused the inactivation of malate dehydrogenase and the aggregation of leghemoglobin. In addition, numerous glycated proteins were identified in vivo, including three key nodule proteins: sucrose synthase, glutamine synthetase, and glutamate synthase. Label-free quantification identified 10 plant proteins and 18 bacterial proteins as age-specifically glycated. Overall, our results suggest that the selective carbonylation or glycation of crucial proteins involved in nitrogen metabolism, transcriptional regulation, and signaling may constitute a mechanism to control cell metabolism and nodule senescence.

Figure 1.

Scheme showing the most common mechanisms of carbonylation and glycation of amino acid residues. A, Polyunsaturated fatty acid (PUFA) oxidation produces unstable lipid hydroperoxides that decompose to secondary products. The Michael addition of the reactive aldehydes to amino acid side chains (mostly His, Cys, and Lys) generates carbonyl derivatives. B, Formation of advanced glycation end products on Arg and Lys residues. HNE, 4-hydroxy-2-nonenal; MDA, malondialdehyde; ONE, 4-oxononenal; P, protein.

Figure 2.

Carbonylation types and sites in the nodule plant proteome. A, Relative abundance (%) of carbonylated amino acid residues. B, Total modifications. C, Modifications found in Lys, His, Cys, and Arg. The percentages were calculated based on the analysis of 238 proteins. In A, the percentage denotes the proportion of each carbonylated residue with respect to the total number of carbonylated residues (377). In B and C, the percentages denote, respectively, the proportion of each modification with respect to the total number of modifications found in 377 sites and the proportion of each modification on individual residues. Acro, Acrolein Michael addition (MA); Croto, crotonaldehyde MA; HHE, 4-hydroxy-2-hexenal MA; HNE, 4-hydroxy-2-nonenal MA; Kox, Lys oxidation to aminoadipic semialdehyde; MDA, malondialdehyde MA; OHE, 4-oxo-2-hexenal MA; ONE, 4-oxo-2-nonenal MA; Pent, pentenal MA; Pox, Pro oxidation to glutamic semialdehyde; Rox, Arg oxidation to glutamic semialdehyde; Tox, Thr oxidation to 2-amino-3-ketobutyric acid.

Figure 3.

Functional classification and predicted subcellular localization of plant carbonylated proteins. A, Number of plant carbonylated proteins in different functional categories. Bean nodule proteins were BLASTed against the Arabidopsis proteome. The closest homologs were classified according to MapMan (http://www.gabipd.de/projects/MapMan). B, Predicted subcellular localization of carbonylated proteins. The subcellular localization of the closest homologs was determined according to the SUBA database (Hooper et al., 2017).

Figure 4.

Effects of MCO on the activity, protein, and carbonylation of MDH. A, MDH activity after 6 h of MCO at different FeCl₃ and ascorbate (ASC) concentrations. CT, Control omitting ASC and FeCl₃; Ox1, 2.5 mm ASC, 10 μm FeCl₃; Ox2, 12.5 mm ASC, 50 μm FeCl₃; Ox3, 25 mm ASC, 100 μm FeCl₃; Ox4, 50 mm ASC, 200 μm FeCl₃; Ox5, 75 mm ASC, 300 μm FeCl₃. Values are means ± se of three replicates. Means denoted by the same letter are not significantly different at P < 0.05 based on Duncan’s multiple range test. B, Coomassie Blue staining of purified MDH after 6 h of MCO at different FeCl₃ and ASC concentrations. Each lane was loaded with 5 μg of protein. C, Effects of MCO on MDH carbonylation. The recombinant protein was subjected to MCO, derivatized (4 μg) with 2,4-dinitrophenylhydrazine, and subjected to SDS-PAGE. Immunoblotting was performed using an anti-dinitrophenylhydrazone antibody. B and C show representative gels and blots, respectively, from three independent experiments.

Figure 5.

Carbonylation of bean nodule Lb. A, Amino acid sequence of Lb in which the carbonylated residues found in vivo are marked with asterisks. B, Immunoblot of purified Lb after 6 h of MCO at different FeCl₃ and ascorbate (ASC) concentrations. CT, Control omitting ASC and FeCl₃; Ox1, 2.5 mm ASC, 10 μm FeCl₃; Ox2, 12.5 mm ASC, 50 μm FeCl₃; Ox3, 25 mm ASC, 100 μm FeCl₃; Ox4, 50 mm ASC, 200 μm FeCl₃; Ox5, 75 mm ASC, 300 μm FeCl₃. C, Immunoblot of purified Lb after 6 h of MCO at different FeCl₃, ASC, and H₂O₂ concentrations. Ox5, 75 mm ASC, 300 μm FeCl₃; Ox6, 75 mm ASC, 300 μm FeCl₃, 1 mm H₂O₂; Ox7, 75 mm ASC, 300 μm FeCl₃, 5 mm H₂O₂. Gels were loaded with 10 μg of protein per lane. D, Immunoblot of bean nodule extracts. Gels were loaded with 50 μg of protein per lane. For B to D, the apparent molecular masses (kD) of the monomer (M), dimer (D), and tetramer (T) are indicated. The anti-Lb antibody and the secondary antibody were used at dilutions of 1:1,000 and 1:40,000, respectively. Blots are representative of at least three independent experiments.

Figure 6.

Carbonylation types and sites in the nodule bacteroid proteome. A, Relative abundance (%) of carbonylated amino acid residues. B, Total modifications. C, Modifications found in Lys, His, Cys, and Arg. The percentages were calculated based on the analysis of 131 proteins. Abbreviations as in Figure 2.

Figure 7.

Functional classification and predicted subcellular localization of bacteroid carbonylated proteins. A, Number of bacteroid carbonylated proteins in different functional categories. The proteins were classified according to the Clusters of Orthologous Groups (COG). B, Predicted subcellular localization of carbonylated proteins according to PSORTb version 3.0 (Yu et al., 2010).

Figure 8.

Relative abundance of AGE-modified sites in plant proteins from nodules at the three developmental stages. A, Total number of AGE-modified sites identified in plant proteins from young (Y), mature (M), and senescent (S) nodules. B, Number of specific AGE classes identified in glycated peptides differentially abundant in mature and senescent nodules relative to young nodules. Abbreviations as in Table 1.

Figure 9.

Label-free quantification of glycated peptides of plant proteins from bean nodules at different developmental stages. A, Gln synthetase PR-1. B, Elongation factor 1-α. The statistical significance of differential expression and glycation was confirmed by the Mann-Whitney U test (means ± se of three to five biological replicates; P < 0.05).

Figure 10.

Relative abundance of AGE-modified sites in bacteroid proteins from nodules at the three developmental stages. A, Total number of AGE-modified sites identified in bacteroid proteins from young (Y), mature (M), and senescent (S) nodules. B, Number of specific AGE classes identified in glycated peptides differentially abundant in mature and senescent nodules relative to young nodules. Abbreviations as in Table 1 and 2.

Figure 11.

Label-free quantification of glycated peptides of bacteroid proteins from bean nodules at different developmental stages. A, Arg biosynthesis bifunctional protein ArgJ. B, α/β-Hydrolase fold protein. The statistical significance of differential expression and glycation was confirmed by the Mann-Whitney U test (means ± se of five biological replicates; P < 0.05).