The natural non-protein amino acid N-β-methylamino-l-alanine (BMAA) is incorporated into protein during synthesis

Abstract

N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

Improved detection of β-N-methylamino-l-alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis)

“…This finding suggests that facile implementation of straightforward depuration as a means of attenuating exposure may not be enough. Moreover, it also supports the idea that there is some kind of bioincorporation occurring within the blue mussel which may or may not be according to the type described by Dunlop [5] and Glover [6] linking incorporation of BMAA to protein synthesis. This brings to the forefront the question as to how BMAA is incorporated or transformed by blue mussels or how it is metabolized in general in various animals.…”

“…It has been established to be produced by widespread diatoms [2] and dinoflagellates [3,4], thus significantly expanding the potential exposure to this neurotoxin. BMAA bioincorporation has been reported to occur through misincorporation during protein synthesis [5,6], partially explaining how it could be biomagnified. According to recent studies, this raises concern about establishing the BMAA presence in food widely consumed by humans [7][8][9].…”

The neurotoxin BMAA in aquatic systems : analysis, occurrence and effects

“…And although precipitated bound BMAA is often referred to as "protein-associated" BMAA, the exact nature of the association of BMAA with proteins in natural tissues also needs to be elucidated. In vitro, BMAA can be incorporated into 1 proteins [65,90], but in vivo experiments with bacteria do not show protein incorporation [91]. Whether protein incorporation also occurs in vivo is therefore still to be determined.…”

“…It was suggested that this latency period was caused by the presence of a 'toxic reservoir', in which BMAA was stored in a protein-associated form and was slowly released in its free form [70]. Recent studies indeed indicate that BMAA can either associate with, or be incorporated into proteins [65,90,141,176], although this association might in some cases be superficial [91]. BMAA can be maternally transferred as shown for mice, either directly [177] or trough milk during lactation [178].…”

“…This fraction is commonly referred to as "protein associated" or "protein bound" [33,35], but the association of BMAA with proteins in natural samples still needs to be elucidated. In vitro, BMAA can be incorporated into proteins and can be associated to proteins through non-covalent bonding [65,90], but in vivo experiments with bacteria do not show protein incorporation [91]. Only few studies look at soluble bound BMAA in an isolated fraction (e.g., [89,92,103,198]).…”