Amino Acids

, Volume 46, Issue 1, pp 199–207

Separate mechanisms for age-related truncation and racemisation of peptide-bound serine

  • Brian Lyons
  • Joanne F. Jamie
  • Roger J. W. Truscott
Original Article

Abstract

Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of l-serine to d-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins.

Keywords

Serine Truncation Racemisation Aging Post translational modification Dehydroalanine 

Abbreviations

O-Me

O-Methyl ether

Ac

Acetyl

Z

Benzyloxy-carbonyl

DHA

Dehydroalanine

Supplementary material

726_2013_1619_MOESM1_ESM.tif (1.5 mb)
Supplementary Figure 1A: Truncation at N-terminal side of Ser as a function of time. Cleavage was calculated based on the amount of Ac-AA formed compared to the amount of each peptide present at time = 0. Peptides were incubated in sodium borate buffer (100 mM, pH 12.5) at 37 °C. (A) = Ac-AASAA and Ac-AAPSAA. (TIFF 1561 kb)
726_2013_1619_MOESM2_ESM.tif (1.5 mb)
Supplementary Figure 1B: Truncation at N-terminal side of Ser as a function of time. Cleavage was calculated based on the amount of Ac-AA formed compared to the amount of each peptide present at time = 0. Peptides were incubated in sodium borate buffer (100 mM, pH 12.5) at 37 °C (B) = Ac-AASAA and Ac-AALSAA.(TIFF 1563 kb)

References

  1. Alexander AJ, Hughes DE (1995) Monitoring of IgG antibody thermal stability by micellar electrokinetic capillary chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 67(20):3626–3632PubMedCrossRefGoogle Scholar
  2. Argirov OK, Lin B, Ortwerth BJ (2004) 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses. J Biol Chem 279(8):6487–6495PubMedCrossRefGoogle Scholar
  3. Bada JL (1972) Kinetics of racemization of amino acids as a function of pH. J Amer Chem Soc 94(4):1371–1373CrossRefGoogle Scholar
  4. Ball LE, Garland DL, Crouch RK, Schey KL (2004) Post-translational modifications of aquaporin 0 (AQP0) in the normal human lens: spatial and temporal occurrence. Biochemistry 43:9856–9865PubMedCrossRefGoogle Scholar
  5. Bongers J, Heimer EP, Lambros T, Pan YCE, Campbell RM, Felix AM (1992) Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor. Int J Pept Protein Res 39(4):364–374PubMedCrossRefGoogle Scholar
  6. Bruckner H, Schafer S, Bahnmuler D, Hausch M (1987) Quantitative determination of the racemization of amino acids in food proteins by chiral-phase capillary gas chromatography. Fresenius’ Zeitschrift für analytische Chemie 327(1):30–31CrossRefGoogle Scholar
  7. Byford MF (1991) Rapid and selective modification of phosphoserine residues catalysed by Ba2+ ions for their detection during peptide microsequencing. Biochem J 280(Pt 1):261–265PubMedGoogle Scholar
  8. Clarke S (1987) Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int J Pept Protein Res 30(6):808–821PubMedCrossRefGoogle Scholar
  9. Cloos PA, Jensen AL (2000) Age-related de-phosphorylation of proteins in dentin: a biological tool for assessment of protein age. Biogerontology 1(4):341–356PubMedCrossRefGoogle Scholar
  10. Cordoba AJ, Shyong B-J, Breen D, Harris RJ (2005) Non-enzymatic hinge region fragmentation of antibodies in solution. J Chromatogr B 818(2):115–121CrossRefGoogle Scholar
  11. D’Angelo MA, Raices M, Panowski SH, Hetzer MW (2009) Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell 136(2):284–295PubMedCentralPubMedCrossRefGoogle Scholar
  12. Dovrat A, Scharf J, Gershon D (1984) Glyceraldehyde 3-phosphate dehydrogenase activity in rat and human lenses and the fate of enzyme molecules in the aging lens. Mech Ageing Dev 28(2–3):187–191PubMedCrossRefGoogle Scholar
  13. Friedrich MG, Lam J, Truscott RJW (2012) Degradation of an old human protein. Age-dependent cleavage of γS crystallin generates a peptide that binds to cell membranes. J Biol Chem 287(46):39012–39020. doi:10.1074/jbc.M112.391565 Google Scholar
  14. Fujii N, Momose Y, Harada K (1996) Kinetic study of racemization of aspartyl residues in model peptides of αA-crystallin. Int J Pept Protein Res 48(2):118–122PubMedCrossRefGoogle Scholar
  15. Fujii N, Takemoto LJ, Matsumoto S, Hiroki K, Boyle D, Akaboshi M (2000) Comparison of aspartic acid content in alpha A-crystallin from normal and age-matched cataractous human lenses. Biochem Biophys Res Commun 278(2):408–413PubMedCrossRefGoogle Scholar
  16. 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(2):785–794PubMedGoogle Scholar
  17. Goodlett DR, Abuaf PA, Savage PA, Kowalski KA, Mukherjee TK, Tolan JW, Corkum N, Goldstein G, Crowther JB (1995) Peptide chiral purity determination: hydrolysis in deuterated acid, derivatization with Marfey’s reagent and analysis using high-performance liquid chromatography–electrospray ionization-mass spectrometry. J Chromatogr A 707(2):233–244PubMedCrossRefGoogle Scholar
  18. Groenen PJTA, van Dongen MJP, Voorter CEM, Bloemendal H, de Jong WW (1993) Age-dependent deamidation of alpha B-crystallin. FEBS Lett 322(1):69–72PubMedCrossRefGoogle Scholar
  19. Hains PG, Truscott RJ (2010) Age-dependent deamidation of lifelong proteins in the human lens. Invest Ophthalmol Vis Sci 51(6):3107–3114PubMedCrossRefGoogle Scholar
  20. Harrington V, McCall S, Huynh S, Srivastava K, Srivastava O (2004) Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses. Mol Vis 10(61):476–489PubMedGoogle Scholar
  21. Hayashi R, Kameda I (1980) Decreased proteolysis of alkali-treated protein: consequences of racemisation in food processing. J Food Sci 45(5):1430–1431CrossRefGoogle Scholar
  22. Heys KR, Friedrich MG, Truscott RJW (2007) Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, α-crystallin, in maintaining lens flexibility. Aging Cell 6(6):807–815PubMedCrossRefGoogle Scholar
  23. Hooi M, Truscott R (2011) Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses. Age 33(2):131–141PubMedCrossRefGoogle Scholar
  24. Hooi MY, Raftery MJ, Truscott RJ (2012a) Age-dependent racemization of serine residues in a human chaperone protein. Protein Sci 22(1):93–100CrossRefGoogle Scholar
  25. Hooi MY, Raftery MJ, Truscott RJ (2012b) Racemization of two proteins over our lifespan: deamidation of asparagine 76 in gammaS crystallin is greater in cataract than in normal lenses across the age range. Invest Ophthalmol Vis Sci 53(7):3554–3561PubMedCrossRefGoogle Scholar
  26. Korlimbinis A, Truscott RJ (2006) Identification of 3-hydroxykynurenine bound to proteins in the human lens. A possible role in age-related nuclear cataract. Biochemistry 45(6):1950–1960. doi:10.1021/bi051744y PubMedCrossRefGoogle Scholar
  27. Kubo T, Kumagae Y, Miller CA, Kaneko I (2003) Beta-amyloid racemized at the Ser26 residue in the brains of patients with Alzheimer disease: implications in the pathogenesis of Alzheimer disease. J Neuropathol Exp Neurol 62(3):248–259PubMedGoogle Scholar
  28. Lampi KJ, Oxford JT, Bachinger HP, Shearer TR, David LL, Kapfer DM (2001) Deamidation of human βB1 alters the elongated structure of the dimer. Exp Eye Res 72(3):279–288PubMedCrossRefGoogle Scholar
  29. Larsen ML, Horder M, Mogensen EF (1990) Effect of long-term monitoring of glycosylated hemoglobin levels in insulin-dependent diabetes mellitus. N Engl J Med 323(15):1021–1025PubMedCrossRefGoogle Scholar
  30. Liu H, Gaza-Bulseco G, Sun J (2006) Characterization of the stability of a fully human monoclonal IgG after prolonged incubation at elevated temperature. J Chromatogr B 837(1–2):35–43CrossRefGoogle Scholar
  31. Lynnerup N, Kjeldsen H, Heegaard S, Jacobsen C, Heinemeier J (2008) Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life. PLoS One 3(1):e1529PubMedCentralPubMedCrossRefGoogle Scholar
  32. Lyons B, Jamie J, Truscott R (2011) Spontaneous cleavage of proteins at serine residues. Int J Pept Res Ther 17(2):1–5CrossRefGoogle Scholar
  33. Masters PM, Bada JL, Samuel Zigler J (1977) Aspartic acid racemisation in the human lens during ageing and in cataract formation. Nature 268(5615):71–73PubMedCrossRefGoogle Scholar
  34. McCudden CR, Kraus VB (2006) Biochemistry of amino acid racemization and clinical application to musculoskeletal disease. Clin Biochem 39(12):1112–1130PubMedCrossRefGoogle Scholar
  35. Miesbauer LR, Zhou X, Yang Z, Sun Y, Smith DL, Smith JB (1994) Post-translational modifications of water-soluble human lens crystallins from young adults. J Biol Chem 269(17):12494–12502PubMedGoogle Scholar
  36. Mills KV, Paulus H (2005) Biochemical mechanisms of intein-mediated protein splicing. Nucleic Acids Mol Biol 16:233–255CrossRefGoogle Scholar
  37. Polgar L (2005) The catalytic triad of serine peptidases. Cell Mol Life Sci 62(19–20):2161–2172PubMedCrossRefGoogle Scholar
  38. Robinson NE, Robinson AB (2001) Deamidation of human proteins. Proc Natl Acad Sci USA 98(22):12409–12413PubMedCrossRefGoogle Scholar
  39. Santhoshkumar P, Udupa P, Murugesan R, Sharma KK (2008) Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation. J Biol Chem 283:8477–8485PubMedCrossRefGoogle Scholar
  40. Savas JN, Toyama BH, Xu T, Yates JR, Hetzer MW (2012) Extremely long-lived nuclear pore proteins in the rat brain. Science 335(6071):942PubMedCentralPubMedCrossRefGoogle Scholar
  41. Sell DR, Monnier VM (2005) Ornithine is a novel amino acid and a marker of arginine damage by oxoaldehydes in senescent proteins. Ann NY Acad Sci 1043:118–128PubMedCrossRefGoogle Scholar
  42. Shapira R, Austin GE, Mirra SS (1988a) Neuritic plaque amyloid in Alzheimer’s disease is highly racemized. J Neurochem 50(1):69–74PubMedCrossRefGoogle Scholar
  43. Shapira R, Wilkinson KD, Shapira G (1988b) Racemization of individual aspartate residues in human myelin basic protein. J Neurochem 50(2):649–654PubMedCrossRefGoogle Scholar
  44. Shapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ (1991) Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of d-aspartate and nuclear weapons-related radiocarbon. J Clin Invest 87(5):1828–1834PubMedCentralPubMedCrossRefGoogle Scholar
  45. Sine HE, Hass LF (1969) Studies on the structure and function of muscle aldolase. IV. The action of dilute alkali on primary structure and its effect on the determination of subunit molecular weight. J Biol Chem 244(2):430–439PubMedGoogle Scholar
  46. Sivan SS, Wachtel E, Tsitron E, Sakkee N, van der Ham F, DeGroot J, Roberts S, Maroudas A (2008) Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid. J Biol Chem 283(14):8796–8801PubMedCrossRefGoogle Scholar
  47. Smith GG, Evans RC (1980) The effect of structure and conditions on the rate of racemization of free and bound amino acids. Biogeochemistry of amino acids. Wiley, NYGoogle Scholar
  48. Srivastava OP (2000) Truncation of lens crystallins. Invest Ophthalmol Vis Sci 41(4):I10Google Scholar
  49. Srivastava OP, Srivastava K (1996) Characterization of three isoforms of a 9 kDa γd-crystallin fragment isolated from human lenses. Exp Eye Res 62(6):593–604PubMedCrossRefGoogle Scholar
  50. Srivastava OP, Srivastava K (2003) Beta B2-crystallin undergoes extensive truncation during aging in human lenses. Biochem Biophys Res Commun 301(1):44–49PubMedCrossRefGoogle Scholar
  51. 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(11):6164–6170PubMedGoogle Scholar
  52. Su SP, McArthur JD, Aquilina JA (2010) Localization of low molecular weight crystallin peptides in the aging human lens using a MALDI mass spectrometry imaging approach. Exp Eye Res 91(1):97–103PubMedCrossRefGoogle Scholar
  53. Su SP, Lyons B, Friedrich M, McArthur JD, Song X, Xavier D, Aquilina JA, Truscott RJ (2012) Molecular signatures of long-lived proteins: autolytic cleavage adjacent to serine residues. Aging Cell 11(6):1125–1127PubMedCrossRefGoogle Scholar
  54. Takemoto LJ (1995) Identification of the in vivo truncation sites at the C-terminal region of alpha-A crystallin from aged bovine and human lens. Curr Eye Res 14(9):837–841PubMedCrossRefGoogle Scholar
  55. Takemoto LJ (1998) Quantitation of specific cleavage sites at the C-terminal region of alpha-A crystallin from human lenses of different age. Exp Eye Res 66(2):263–266PubMedCrossRefGoogle Scholar
  56. Truscott RJW (2010) Are ancient proteins responsible for the age-related decline in health and fitness? Rejuvenation Res 13:83–89. doi:10.1089/rej.2009.0938 PubMedCrossRefGoogle Scholar
  57. Truscott RJ, Mizdrak J, Friedrich MG, Hooi MY, Lyons B, Jamie JF, Davies MJ, Wilmarth PA, David LL (2012) Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine? Exp Eye Res 99:48–54PubMedCentralPubMedCrossRefGoogle Scholar
  58. Wang Z, Lyons B, Truscott RJW, Schey KL (2013) Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates. Aging Cell. doi:10.1111/acel.12164
  59. Wilmarth PA, Tanner S, Dasari S, Nagalla SR, Riviere MA, Bafna V, Pevzner PA, David LL (2006) Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility? J Proteome Res 5(10):2554–2566PubMedCentralPubMedCrossRefGoogle Scholar
  60. Yamasaki M, Takahashi N, Hirose M (2003) Crystal structure of S-ovalbumin as a non-loop-inserted thermostabilized serpin form. J Biol Chem 278(37):35524–35530PubMedCrossRefGoogle Scholar
  61. Zhu X, Korlimbinis A, Truscott RJW (2010) Age-dependent denaturation of enzymes in the human lens: a paradigm for organismic aging? Rejuvenation Res 13:553–560PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Brian Lyons
    • 1
  • Joanne F. Jamie
    • 2
  • Roger J. W. Truscott
    • 3
  1. 1.Save Sight Institute, Sydney Eye HospitalUniversity of SydneySydneyAustralia
  2. 2.Department of Chemistry and Biomolecular SciencesMacquarie UniversitySydneyAustralia
  3. 3.Illawarra Health and Medical Research InstituteUniversity of WollongongWollongongAustralia

Personalised recommendations