Azetidine-2-carboxylic acid (A2C) is a non-proteinogenic amino acid and four-membered ring analogue of proline found in plants of the Beta vulgaris family, including sugar beet. A2C is mistakenly incorporated into proteins in place of proline, leading to structural and functional alterations. While its role in protein misfolding and antigenicity has been suggested, the proteome-wide consequences of A2C incorporation remain unclear. Here, we exposed human neuroblastoma (SH-SY5Y) cells to A2C (500 µM) for 24 hours and identified 226 A2C-containing proteins by mass spectrometry. These proteins were enriched in pathways involving protein folding, the unfolded protein response (UPR), and antigen processing. The presence of exogenous proline reduced A2C incorporation by >92%, highlighting competition at the translational level.
Proteomic analysis of total protein abundance revealed a downregulation in translation initiation, gene expression, and metabolic pathways, consistent with a cellular response to misfolded proteins. A2C-containing proteins were also enriched in extracellular exosome pathways, suggesting a mechanism for their export and immune recognition. Furthermore, proteins associated with systemic lupus erythematosus (SLE) and neurodegenerative diseases were significantly enriched, supporting broader autoimmune implications.
Given the increasing use of sugar beet-derived products as an alternative to cane sugar, human exposure to A2C may be widespread. Epidemiological studies linking high sugar beet consumption to increased multiple sclerosis (MS) incidence raise the possibility that A2C incorporation into myelin basic protein (MBP) generates neoantigens triggering autoimmunity. The co-enrichment of SLE-associated proteins further suggests a role for A2C in multiple autoimmune disorders.
These findings provide evidence for widespread A2C incorporation into the proteome and highlight the need for further investigation into its long-term impact on human health, particularly MS, SLE, and neurodegenerative diseases.