Investigating β-N-Methylamino-l-alanine Misincorporation in Human Cell Cultures: A Comparative Study with Known Amino Acid Analogues

Abstract

Misincorporation of β-N-methylamino-l-alanine (BMAA) into proteins has been proposed to be a mechanism of toxicity to explain the role of BMAA in neurodegenerative disease development. However, studies have shown that all detectable BMAA can be removed from proteins by SDS-PAGE purification and that the toxicity of l-canavanine cannot be reproduced in prokaryotes or in a rat pheochromocytoma cell line, strongly indicating that the misincorporation hypothesis of BMAA should be re-investigated. The aim of this study was therefore to determine if BMAA misincorporates into proteins in cells of human origin with subsequent misincorporation-type toxicity. Almost complete loss of viability in response to exposure to l-4-fluorophenylalanine and l-m-tyrosine was observed in all of the cell lines, corresponding to a concentration-dependent increase of the analogues in protein extracts from exposed cells. In contrast, BMAA exposure resulted in slight toxicity in one of the cell lines but the observed toxicity was not the result of misincorporation of BMAA into proteins, as no BMAA was detected in any of the SDS-PAGE purified protein extracts that were obtained from the cells following BMAA exposure. The results show that BMAA is not misincorporated into human proteins and that misincorporation is not a valid mechanism of toxicity.

Occurrence of β-N-methylamino-l-alanine (BMAA) and Isomers in Aquatic Environments and Aquatic Food Sources for Humans

“…To date, the precursors giving BMAA after hydrolysis of protein or polypeptide fractions are still unknown [ 21 ], as well as the exact nature of the interaction between BMAA and proteins or peptides. The mechanism of BMAA association to proteins, through non-specific or non-covalent bonding, or its covalent misincorporation during the protein synthesis causing protein misfolding, aggregation and/or loss of function, have been both suggested but not clearly proved or disproved [ 9 , 66 ] and remains debated [ 11 ]. The results of an exposure of various freshwater mussels to dissolved labeled 5+ BMAA suggested a metabolization via a reversible covalent modification of BMAA, variable in different species, but did not provide any evidence of BMAA association with proteins [ 64 ].…”

“…BMAA has been suggested to bind to or incorporate into proteins during their synthesis, which could induce protein misfolding and cell dysfunction [ 9 ]. However, this hypothesis has been criticized and BMAA may only be chemically associated to proteins rather than integrated into [ 10 , 11 ].…”

Microbial BMAA and the Pathway for Parkinson’s Disease Neurodegeneration

“…While the observation that BMAA interacts very strongly with proteins causing their misfolding has not been disputed, some groups have failed to detect incorporation of BMAA into the primary structure of proteins thus calling into question the role of the misincorporation hypothesis in explaining BMAA's observed neurotoxic effects (Beri et al, 2017;Onselen et al, 2017). Subsequently, alternative mechanisms have been investigated and BMAA has been found to strongly associate with melanin, to selectively inhibit the activity of certain enzymes and to interfere with and disrupt protein refolding in vitro by associating with proteins through electrostatic interactions strong enough to resist TCA precipitation and subsequent washing with SDS or DTT (van Onselen and Downing, 2018).…”

From Superficial Damage to Invasion of the Nucleosome: Ranking of Morbidities by the Biosemiotic Depth Hypothesis

“…At the level of proteinogenesis, some researchers have argued that non-coding amino acids such as beta-methylamino-L-alanine (BMAA, similar to serine; [240]- [244], canavanine (similar to arginine; [240], [241], [245], and glypohosate (similar to glycine; [145], [246], [247] can lead to erroneous interpretations and are, in some instances, misincorporated into necessary proteins [148], [248] resulting in deformed strings that lead to disease conditions including systemic autoimmunities [245], [249]- [252]. However, the relevant research, at least in the case of BMAA shows that wholesale misincorporation of non-protoeinogenic amino acids is not easily achieved in living cells [253], [254]. BMAA misincorporation is resisted even in bacteria [242], though misincorporation can be humanly engineered and achieved efficiently in vitro under special conditions [251], [255], [256].…”

“…It is still uncertain when and where misincorporation can occur in vivo, though some research suggests that misincorporation of canavanine in place of arginine may happen in the mitochondrial ribosome causing normal protein synthesis to stall [257]. Because misincorporation of BMAA, for one, is blocked in many cases by editing taking place in the mitochoindria [258], [259], a minor level of excitotoxicity of non-coding BMAA, rather than its misincorporation into proteins [243], [254], is believed by some to be causing cell death and contributing to systemic autoimmunities [260], [261].…”