A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues
- 552 Downloads
The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day−1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k D2O/k H2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.
KeywordsAlzheimer’s disease PIMT Capillary electrophoresis 1H NMR Isoaspartate
We are grateful to Prof. Dr. H.-U. Demuth for his helpful comments and suggestions on the manuscript. We thank Dr. T. Hoffmann for his technical support. This work was financially supported by the Investitionsbank Sachsen-Anhalt, Grant# 1004/00082 to Probiodrug AG (StS).
Conflict of interest
BG, HC, FS, H-HL and StS are former or present employees of Probiodrug AG.
- Capasso S, Mazzarella L, Sica F, Zagari A, Salvadori S (1993) Kinetics and mechanism of succinimide ring formation in the deamidation process of asparagine residues. J Chem Soc Perkin Trans 2:679–682Google Scholar
- Cook PF (1991) Enzyme mechanism from isotope effects. CRC press, Boca RatonGoogle Scholar
- Di Fede G, Catania M, Morbin M, Rossi G, Suardi S, Mazzoleni G, Merlin M, Giovagnoli AR, Prioni S, Erbetta A, Falcone C, Gobbi M, Colombo L, Bastone A, Beeg M, Manzoni C, Francescucci B, Spagnoli A, Cantù L, Del Favero E, Levy E, Salmona M, Tagliavini F (2009) A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis. Science 323:1473–1477Google Scholar
- Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, Hoyte K, Gustafson A, Liu Y, Lu Y, Bhangale T, Graham RR, Huttenlocher J, Bjornsdottir G, Andreassen OA, Jonsson EG, Palotie A, Behrens TW, Magnusson OT, Kong A, Thorsteinsdottir U, Watts RJ, Stefansson K (2012) A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488:96–99PubMedCrossRefGoogle Scholar
- Nussbaum JM, Schilling S, Cynis H, Silva A, Swanson E, Wangsanut T, Tayler K, Wiltgen B, Hatami A, Ronicke R, Reymann K, Hutter-Paier B, Alexandru A, Jagla W, Graubner S, Glabe CG, Demuth HU, Bloom GS (2012) Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-beta. Nature 485:651–655PubMedCrossRefGoogle Scholar
- Park JW, Lee JC, Ha SW, Bang SY, Park EK, Yi SA, Lee MG, Kim DS, Nam KH, Yoo JH, Kwon SH, Han JW (2012) Requirement of protein l-isoaspartyl O-methyltransferase for transcriptional activation of trefoil factor 1 (TFF1) gene by estrogen receptor alpha. Biochem Biophys Res Commun 420:223–229PubMedCrossRefGoogle Scholar
- Roher AE, Lowenson JD, Clarke S, Wolkow C, Wang R, Cotter RJ, Reardon IM, Zurcher-Neely HA, Heinrikson RL, Ball MJ (1993) Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer’s disease. J Biol Chem 268:3072–3083PubMedGoogle Scholar
- Schilling S, Zeitschel U, Hoffmann T, Heiser U, Francke M, Kehlen A, Holzer M, Hutter-Paier B, Prokesch M, Windisch M, Jagla W, Schlenzig D, Lindner C, Rudolph T, Reuter G, Cynis H, Montag D, Demuth HU, Rossner S (2008) Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer’s disease-like pathology. Nat Med 14:1106–1111PubMedCrossRefGoogle Scholar
- Wagenknecht H-A, Knapp H (2009) Roempp Online. Version 3.5Google Scholar