Amino Acids

, Volume 44, Issue 4, pp 1205–1214 | Cite as

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

  • Bert H.-O. Güttler
  • Holger Cynis
  • Franziska Seifert
  • Hans-Henning Ludwig
  • Andrea Porzel
  • Stephan Schilling
Original Article


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.


Alzheimer’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.


  1. Aswad DW, Paranandi MV, Schurter BT (2000) Isoaspartate in peptides and proteins: formation, significance, and analysis. J Pharm Biomed Anal 21:1129–1136PubMedCrossRefGoogle Scholar
  2. Barnett RE, Jencks WP (1969) Diffusion-controlled proton transfer in intramolecular thiol ester aminolysis and thiazoline hydrolysis. J Am Chem Soc 91:2358–2369CrossRefGoogle Scholar
  3. Böhme L, Bär JW, Hoffmann T, Manhart S, Ludwig HH, Rosche F, Demuth HU (2008a) Isoaspartate residues dramatically influence substrate recognition and turnover by proteases. Biol Chem 389:1043–1053PubMedGoogle Scholar
  4. Böhme L, Hoffmann T, Manhart S, Wolf R, Demuth HU (2008b) Isoaspartate containing amyloid precursor protein derived peptides alter efficacy and specificity of potential beta-secretases. Biol. Chem. 389:1055–1066PubMedGoogle Scholar
  5. 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
  6. Carter WG, Aswad DW (2008) Formation, localization, and repair of L-isoaspartyl sites in histones H2A and H2B in nucleosomes from rat liver and chicken erythrocytes. Biochemistry 47:10757–10764PubMedCrossRefGoogle Scholar
  7. Clarke S (1985) Protein carboxyl methyltransferases: two distinct classes of enzymes. Annu Rev Biochem 54:479–506PubMedCrossRefGoogle Scholar
  8. Cook PF (1991) Enzyme mechanism from isotope effects. CRC press, Boca RatonGoogle Scholar
  9. Corti A, Curnis F (2011) Isoaspartate-dependent molecular switches for integrin-ligand recognition. J Cell Sci 124:515–522PubMedCrossRefGoogle Scholar
  10. Cox MM, Jencks WP (1978) General acid catalysis of the aminolysis of phenyl acetate by a preassociation mechanism. J Am Chem Soc 100:5956–5957CrossRefGoogle Scholar
  11. 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
  12. Ellis KJ, Morrison JF (1982) Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol 87:405–426PubMedCrossRefGoogle Scholar
  13. Geiger T, Clarke S (1987) Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem 262:785–794PubMedGoogle Scholar
  14. George-Nascimento C, Lowenson J, Borissenko M, Calderon M, Medina-Selby A, Kuo J, Clarke S, Randolph A (1990) Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor. Biochemistry 29:9584–9591PubMedCrossRefGoogle Scholar
  15. Haass C, Lemere CA, Capell A, Citron M, Seubert P, Schenk D, Lannfelt L, Selkoe DJ (1995) The Swedish mutation causes early-onset Alzheimer’s disease by beta-secretase cleavage within the secretory pathway. Nat Med 1:1291–1296PubMedCrossRefGoogle Scholar
  16. He W, Barrow CJ (1999) The A beta 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater beta-sheet forming and aggregation propensities in vitro than full-length A beta. Biochemistry 38:10871–10877PubMedCrossRefGoogle Scholar
  17. 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
  18. Kummer MP, Hermes M, Delekarte A, Hammerschmidt T, Kumar S, Terwel D, Walter J, Pape HC, König S, Roeber S, Jessen F, Klockgether T, Korte M, Heneka MT (2011) Nitration of tyrosine 10 critically enhances amyloid β aggregation and plaque formation. Neuron 71:833–844PubMedCrossRefGoogle Scholar
  19. Lee JC, Kang SU, Jeon Y, Park JW, You JS, Ha SW, Bae N, Lubec G, Kwon SH, Lee JS, Cho EJ, Han JW (2012) Protein l-isoaspartyl methyltransferase regulates p53 activity. Nat. Commun. 3:927PubMedCrossRefGoogle Scholar
  20. Lowenson JD, Clarke S (1990) Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase. J Biol Chem 265:3106–3110PubMedGoogle Scholar
  21. Lowenson JD, Clarke S (1991) Structural elements affecting the recognition of l-isoaspartyl residues by the l-isoaspartyl/d-aspartyl protein methyltransferase. Implications for the repair hypothesis. J Biol Chem 266:19396–19406PubMedGoogle Scholar
  22. Lowenson JD, Clarke S (1992) Recognition of d-aspartyl residues in polypeptides by the erythrocyte l-isoaspartyl/d-aspartyl protein methyltransferase. Implications for the repair hypothesis. J Biol Chem 267:5985–5995PubMedGoogle Scholar
  23. Lowenson JD, Roher AE, Clarke S (1994) Protein aging extracellular amyloid formation and intracellular repair. Trends Cardiovasc Med 4:3–8PubMedCrossRefGoogle Scholar
  24. MacLaren DC, Kagan RM, Clarke S (1992) Alternative splicing of the human isoaspartyl protein carboxyl methyltransferase RNA leads to the generation of a C-terminal -RDEL sequence in isozyme II. Biochem Biophys Res Commun 185:277–283PubMedCrossRefGoogle Scholar
  25. Meinwald YC, Stimson ER, Scheraga HA (1986) Deamidation of the asparaginyl-glycyl sequence. Int J Pept Protein Res 28:79–84PubMedCrossRefGoogle Scholar
  26. Ni W, Dai S, Karger BL, Zhou ZS (2010) Analysis of isoaspartic acid by selective proteolysis with Asp-N and electron transfer dissociation mass spectrometry. Anal Chem 82:7485–7491PubMedCrossRefGoogle Scholar
  27. Noguchi S (2010) Conformational variation revealed by the crystal structure of RNase U2A complexed with Ca ion and 2′-adenylic acid at 1.03 Å resolution. Protein Pept Lett 17:1559–1561PubMedCrossRefGoogle Scholar
  28. 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
  29. 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
  30. Patel K, Borchardt RT (1990) Chemical pathways of peptide degradation. II. Kinetics of deamidation of an asparaginyl residue in a model hexapeptide. Pharm Res 7:703–711PubMedCrossRefGoogle Scholar
  31. Potter SM, Johnson BA, Henschen A, Aswad DW, Guzzetta AW (1992) The type II isoform of bovine brain protein l-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (…RDEL) at its C-terminus. Biochemistry 31:6339–6347PubMedCrossRefGoogle Scholar
  32. Robinson AB (1974) Evolution and the distribution of glutaminyl and asparaginyl residues in proteins. Proc Natl Acad Sci USA 71:885–888PubMedCrossRefGoogle Scholar
  33. Robinson NE, Robinson AB (2001) Molecular clocks. Proc Natl Acad Sci USA 98:944–949PubMedCrossRefGoogle Scholar
  34. Robinson AB, Rudd CJ (1974) Deamidation of glutaminyl and asparaginyl residues in peptides and proteins. Curr Top Cell Regul 8:247–295PubMedGoogle Scholar
  35. Robinson AB, McKerrow JH, Cary P (1970) Controlled deamidation of peptides and proteins: an experimental hazard and a possible biological timer. Proc Natl Acad Sci USA 66:753–757PubMedCrossRefGoogle Scholar
  36. 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
  37. Saido TC, Yamao-Harigaya W, Iwatsubo T, Kawashima S (1996) Amino- and carboxyl-terminal heterogeneity of beta-amyloid peptides deposited in human brain. Neurosci Lett 215:173–176PubMedCrossRefGoogle Scholar
  38. Sargaeva NP, Lin C, O’Connor PB (2011) Differentiating N-terminal aspartic and isoaspartic acid residues in peptides. Anal Chem 83:6675–6682PubMedCrossRefGoogle Scholar
  39. Satterthwait AC, Jencks WP (1974) The mechanism of the aminolysis of acetate esters. J Am Chem Soc 96:7018–7031PubMedCrossRefGoogle Scholar
  40. Schilling S, Hoffmann T, Manhart S, Hoffmann M, Demuth HU (2004) Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions. FEBS Lett 563:191–196PubMedCrossRefGoogle Scholar
  41. Schilling S, Lauber T, Schaupp M, Manhart S, Scheel E, Bohm G, Demuth HU (2006) On the seeding and oligomerization of pGlu-amyloid peptides (in vitro). Biochemistry 45:12393–12399PubMedCrossRefGoogle Scholar
  42. 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
  43. Schlenzig D, Manhart S, Cinar Y, Kleinschmidt M, Hause G, Willbold D, Funke SA, Schilling S, Demuth HU (2009) Pyroglutamate formation influences solubility and amyloidogenicity of amyloid peptides. Biochemistry 48:7072–7078PubMedCrossRefGoogle Scholar
  44. Seifert F, Schulz K, Koch B, Manhart S, Demuth HU, Schilling S (2009) Glutaminyl cyclases display significant catalytic proficiency for glutamyl substrates. Biochemistry 48:11831–11833PubMedCrossRefGoogle Scholar
  45. Shimizu T, Watanabe A, Ogawara M, Mori H, Shirasawa T (2000) Isoaspartate formation and neurodegeneration in Alzheimer’s disease. Arch Biochem Biophys 381:225–234PubMedCrossRefGoogle Scholar
  46. Stephenson RC, Clarke S (1989) Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J Biol Chem 264:6164–6170PubMedGoogle Scholar
  47. Varshavsky A (1996) The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci USA 93:12142–12149PubMedCrossRefGoogle Scholar
  48. Velazquez P, Cribbs DH, Poulos TL, Tenner AJ (1997) Aspartate residue 7 in amyloid beta-protein is critical for classical complement pathway activation: implications for Alzheimer’s disease pathogenesis. Nat Med 3:77–79PubMedCrossRefGoogle Scholar
  49. Wagenknecht H-A, Knapp H (2009) Roempp Online. Version 3.5Google Scholar
  50. Weber DJ, McFadden PN (1997) Injury-induced enzymatic methylation of aging collagen in the extracellular matrix of blood vessels. J Protein Chem 16:269–281PubMedCrossRefGoogle Scholar
  51. Young AL, Carter WG, Doyle HA, Mamula MJ, Aswad DW (2001) Structural integrity of histone H2B in vivo requires the activity of protein l-isoaspartate O-methyltransferase, a putative protein repair enzyme. J Biol Chem 276:37161–37165PubMedCrossRefGoogle Scholar
  52. Young GW, Hoofring SA, Mamula MJ, Doyle HA, Bunick GJ, Hu Y, Aswad DW (2005) Protein l-isoaspartyl methyltransferase catalyzes in vivo racemization of aspartate-25 in mammalian histone H2B. J Biol Chem 280:26094–26098PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Bert H.-O. Güttler
    • 1
  • Holger Cynis
    • 1
  • Franziska Seifert
    • 1
  • Hans-Henning Ludwig
    • 1
  • Andrea Porzel
    • 2
  • Stephan Schilling
    • 1
  1. 1.Probiodrug AGHalle (Saale)Germany
  2. 2.Department of Bioorganic ChemistryLeibniz Institute of Plant BiochemistryHalle (Saale)Germany

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