title: 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.
authors.affiliation: Department of Chemistry, University of British Columbia, 3247 University Way, Kelowna, BC, V1V 1V7, Canada.
authors.forename: W Broc
authors.initials: WB
authors.lastname: Glover
authors.forename: Deborah C
authors.initials: DC
authors.lastname: Mash
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Murch
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000596:Amino Acids / Q000737:chemistry*; D000599:Amino Acids, Diamino / Q000737:chemistry*; D000613:Aminobutyrates / Q000737:chemistry; D001921:Brain / Q000473:pathology; D002474:Cell-Free System; D002853:Chromatography, Liquid; D000087522:Cyanobacteria Toxins; D004247:DNA / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008297:Male; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D000599:Amino Acids, Diamino; D000613:Aminobutyrates; D000087522:Cyanobacteria Toxins; D011506:Proteins; C001824:beta-N-methylamino-L-alanine; C028791:N-(2-aminoethyl)glycine; C005959:2,4-diaminobutyric acid; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D004229:Dithiothreitol; D005998:Glycine