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 () 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.

Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?

“…Thereby, in comparison to the mammalian proteins, plant polypeptides demonstrated much higher numbers of AGE sites, where modifications were formed mostly via oxidative glycosylation [25]. Moreover, a significant increase of glycation levels at specific protein residues was characteristic for ageing of Arabidopsis leaves [26] and pea root nodules [27]. Not less importantly, enhanced AGE formation accompanied environmental stress [28].…”

Proteome Map of Pea (Pisum Sativum L.) Embryos Containing Different Amounts of Residual Chlorophylls

“…ultra filtration [38] or pre-separation by PAGE [39]). Finally, our sequence database, containing protein entries of three legumes, related to pea, already proved to be a reliable tool for the identification of low-abundant post-translationally modified (glycated) tryptic peptides (known to yield complex fragmentation [40]) in common bean [41]. The database contained exclusively reviewed (i.e.…”

“…Each time, the samples were centrifuged (5000 g, 10 min, 4° C) after re-suspension. Finally, the cleaned pellets were dried under air flow in a fume hood for 1 h, reconstituted in 100 µL of shotgun buffer (8 mol/L urea, 2 mol/L thiourea, 0.15% AALS II in 100 mmol/L tris-HCl, pH 7.5), and protein contents were determined by 2-D Quant Kit (GE Healthcare, Taufkirchen, Germany) according to Matamoros and co-workers [41] or by the Bradford method as described by Frolov and co-workers [71]. The precision of the assay was verified by SDS-PAGE according Greifenhagen et al [72] with minor modifications (for details see Protocol S1-4).…”

“…The mass spectrometer settings and DDA parameters are summarized in Table S1-2. The database search relied on Proteome Discoverer 2.2 software (Thermo Fisher Scientific, Bremen, Germany), SEQUEST search engine and a combined sequence database comprising proteomes of MedicagotruncatulaGaertn, Lotus japonicus (Regel) K. Larsen and Phaseolus vulgaris L [41] with addition of protein sequences, prospectively related to seed longevity and color (see Table S1-3 for details). The redundancy of the database was eliminated by the СD-HIT algorithm [73] with the sequence identity cut-off set to 1.…”

Analysis of Chemically Labile Glycation Adducts in Seed Proteins: Case Study of Methylglyoxal-Derived Hydroimidazolone 1 (MG-H1)

“…Moreover, glycation of specific proteins was enhanced not only in presence of environmental stressors, like drought [19], but also accompanied ageing in leaves [20], legume nodules [21] and seeds [22]. 2-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3) [24].…”