TMT-based quantitative proteomic assessment of Vicia sativa induced neurotoxicity by β-cyano-L-alanine and γ-glutamyl-β-cyano-L-alanine in SH-SY5Y cells.

Abstract

{beta}-Cyano-L-alanine (BCA) and {gamma}-glutamyl-{beta}-cyano-L-alanine (GBCA) are the primary antinutritional compounds present within the seeds of the high protein and drought tolerant orphan legume Vicia sativa. Evidence of neurotoxicity is limited to symptom analysis from animal feed trials whilst the molecular mechanisms underlying suspected neurotoxicity in monogastric animals are largely unknown. In this study, we first optimised an in vitro cell-based assay for rapid testing of BCA and GBCA toxicity in the retinoic acid differentiated SH-SY5Y human neuroblastoma cells. Using this system, we then performed proteomics analyses to determine dysregulated expression of proteins in BCA or GBCA treated of differentiated SH-SY5Y cells. Our findings indicate that BCA affects expression of proteins involved in DNA damage and translation whilst GBCA treatment causes dysregulation of those involved in mitosis and cell cycle. Following BCA treatment, we identified changes in many proteins previously suggested to have close association with neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimers disease as well as cancers. Following GBCA treatment, dysregulation of proteins involved in apoptosis pathways was observed. Finally, the lack of common dysregulated proteins and pathways in BCA or GBCA treated cells indicated that they most likely cause neurotoxicity via distinct mechanisms. SignificanceAntinutritional compounds or toxins limit the use of many potential grain crops including V. sativa (Common Vetch), and BCA and GBCA are the two principal toxic compounds in Common Vetch grain. Vetch grain toxicity is associated with neurotoxic symptoms in animals. Characterizing the proteome of BCA- and GBCA-treated neural cells gives insights into the excitotoxicity mechanism and identification of biomarkers for screening higher quality grain. Quantitative tandem mass tag (TMT) mass spectrometry-based proteome profiling of BCA treated neural cells characterized 6,827 proteins, of which 26 were significantly up-regulated and 73 significantly downregulated. However, TMT proteome analysis of GBCA treated cells identified 76 significantly up regulated and 86 downregulated proteins. These results provide insight into the toxicity of BCA and GBCA and enable future identification of putative direct protein targets. We describe for the first time BCA playing a role in modulating the expression DNA damage proteins and translation, and GBCA playing a role in modulating the expression of mitosis and cell cycle proteins, elucidating the mechanisms of plant-derived toxins in mammalian cell neurotoxicity HighlightsO_LIEstablished in vitro cell-based toxicity assay for {beta}-cyano-L-alanine and {gamma}-glutamyl-{beta}-cyano-L-alanine. C_LIO_LI{beta}-Cyano-L-alanine treatment dysregulated many proteins associated with DNA damage in retinoic acid differentiated SH-SY5Y cells. C_LIO_LI{gamma}-Glutamyl-{beta}-cyano-L-alanine treatment dysregulated proteins associated with cell cycle. C_LI