In vitro and in vivo characterization of plant-specific glycation products in terms of their structure, origin and effects on mammalian cells

• Applicant: Dr. Andrej Frolov
• Subject Area: Food Chemistry
• Term: from 2012 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 224903464

• Final Report Year: 2024

Final Report Abstract

To summarize, in general, the project can be treated as successful. The peptide-based glycation model once more proved its high potential for producing informative results about site-specific glycation patterns. The obtained time courses for highly-reactive plant derived glycation agents delivered valuable information for future alignment of in vitro and in vivo data. We also got primary data on the patterns of site specific lipoxidation. Overall, quite large numbers of adducts could be annotated, but before investing time in their identification and preparative synthesis we addressed the question which of them are drought-regulated. Surprisingly, even in such a protein-rich plant part as seeds we could not find any quantitative differences for the well-known protein glycation adducts. This failure was unexpected, and it raised the question about the compatibility of all setups – peptide models, proteomics and absolute quantification of specific AGEs for both in vitro and in vivo data. To clear this issue, we decided to transfer our peptide-based model to the in planta level using a molecular biology approach. In terms of this approach, our model sequence was selectively expressed in chloroplasts of N. bentamiana leaves as a part of transiently expressed GFP-based construct. Remarkably, selection of chloroplasts as the expression site was not random. Indeed, the Calvin cycle reactions are localized their, i.e. the most reactive plant glycation agents (according our previous results) are localized to these organelles. Moreover, according to the acquired time courses, the anticipated amid AGEs (the major products of glycation with ribulose-1,5-diphospate and its triosophosphate metabolites) required only 3-5 days to be formed. It ideally suited to the prove-of the concept transient expression experiment. Fortunately, our hypothesis worked, and it is definitely the main output of the project. Moreover, we got stable transformants, which were successfully selected, reproduced and now we are going set up ageing and drought stress experiments with these plants. With this extension and with up-scaling of the enrichment step we hope to get complete patterns of glycation products and to give the final answers to all questions set. Here is important to note, that all required methodology for the biological activity tests is optimized and is ready to use when the in planta patterns are fully characterized. When the ageing and stress-related data will be acquired, the manuscript with these break-through concept will be submitted.

Publications

• Analysis of Chemically Labile Glycation Adducts in Seed Proteins: Case Study of Methylglyoxal-Derived Hydroimidazolone 1 (MG-H1). International Journal of Molecular Sciences, 20(15), 3659.
  Antonova, Kristina; Vikhnina, Maria; Soboleva, Alena; Mehmood, Tahir; Heymich, Marie-Louise; Leonova, Tatiana; Bankin, Mikhail; Lukasheva, Elena; Gensberger-Reigl, Sabrina; Medvedev, Sergei; Smolikova, Galina; Pischetsrieder, Monika & Frolov, Andrej
  
• Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?. International Journal of Molecular Sciences, 21(2), 567.
  Leonova, Tatiana; Popova, Veronika; Tsarev, Alexander; Henning, Christian; Antonova, Kristina; Rogovskaya, Nadezhda; Vikhnina, Maria; Baldensperger, Tim; Soboleva, Alena; Dinastia, Ekaterina; Dorn, Mandy; Shiroglasova, Olga; Grishina, Tatiana; Balcke, Gerd U.; Ihling, Christian; Smolikova, Galina; Medvedev, Sergei; Zhukov, Vladimir A.; Babakov, Vladimir ... & Frolov, Andrej
  
• Does filter-aided sample preparation provide sufficient method linearity for quantitative plant shotgun proteomics?. Frontiers in Plant Science, 13.
  Leonova, Tatiana; Ihling, Christian; Saoud, Mohamad; Frolova, Nadezhda; Rennert, Robert; Wessjohann, Ludger A. & Frolov, Andrej
  
• Dynamics of Reactive Carbonyl Species in Pea Root Nodules in Response to Polyethylene Glycol (PEG)-Induced Osmotic Stress. International Journal of Molecular Sciences, 23(5), 2726.
  Soboleva, Alena; Frolova, Nadezhda; Bureiko, Kseniia; Shumilina, Julia; Balcke, Gerd U.; Zhukov, Vladimir A.; Tikhonovich, Igor A. & Frolov, Andrej