Post-translational methylation of asparaginyl residues. Identification of beta-71 gamma-N-methylasparagine in allophycocyanin.

Abstract

A novel post-translationally modified residue, gamma-N-methylasparagine, was detected in the beta subunit of Anabaena variabilis allophycocyanin. Structure determination was accomplished by isolating a decapeptide, AP-beta (63-72) shown to have the following structure: Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly- Asn[N-CH3]-homoserine lactone Fast atom bombardment-mass spectrometry established that the residue corresponding to position 71 in the protein (DeLange, R. J., Williams, L. C., and Glazer, A. N. (1981) J. Biol. Chem. 256, 9558-9566) contained 13 mass units more than expected for aspartic acid though aspartic acid was recovered after acid hydrolysis. The 1H NMR spectrum of AP-beta (63-72) revealed a strong methyl single at 2.71 ppm characteristic of the methyl derivative of an amide nitrogen. Confirmation of this bond arrangement was obtained by detection of a stoichiometric amount of methylamine in acid hydrolysates of the peptide. This is the first report of gamma-N-methylasparagine in a protein. Amino acid analysis of A. variabilis allophycocyanin subunits showed that the derivative at position 71 can account for the total methylamine released from the beta subunit, while hydrolysis of the alpha subunit released no methylamine. The beta subunits of the allophycocyanins from the cyanobacterium Synechococcus PCC 6301 and the red alga Porphyridium cruentum each released 1 eq of methylamine upon acid hydrolysis. No methylamine was released from the alpha subunits.