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Characterization of N-homocysteinylated Albumin Adducts

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Abstract

Increased plasma homocysteine levels are considered an important risk factor for vascular disease. Homocysteine, an intermediate compound in methionine metabolism, is an amino acid that includes a thiol group, and circulates as different species. One of them, Homocysteine thiolactone (HTL) forms adducts through irreversible reactions with epsilon-NH2 groups of lysine residues. These processes can alter the structure and biological function of diverse proteins that may be involved in the detrimental effects of homocysteine. Particularly, in this work we evaluated HTL-mediated molecular changes in human serum albumin (HSA) through electrophoretic techniques. Albumin and HTL were incubated (37 °C, 6 h) at HSA:HTL molar ratios of 1:25, 1:50 and 1:100. Polyacrylamid gel electrophoresis showed that electrophoretic mobility was increased in the treated HSA respect to control, in an HTL-concentration-dependent manner. That anodic shift of the treated samples was also observed in crossed immunoelectrophoresis profiles. As expected, a decrease in the isoelectric point of the homocysteinylated albumin (pI 4.7) in comparison to that of control (pI 4.8) was shown by the isoelectric focusing technique. Moreover, the electropherogram acquired by capillary zone electrophoresis indicated that migration times and full width at half height were enhanced with the rise of HTL concentration. We propose that the in vitro structural changes of albumin described in the present work would be involved in the harmful effects of the N-homocysteinylation process.

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Abbreviations

CIE:

Crossed immunoelectrophoresis

CZE:

Capillary zone electrophoresis

IEF:

Isoelectric focusing

Hcy:

Homocysteine

tHcy:

Total homocysteine

HSA:

Human serum albumin

HTL:

Homocysteine thiolactone

HTL-N-HSA:

N-homocysteinylated human serum albumin

PAGE:

Polyacrylamide gel electrophoresis

References

  1. Homocysteine Studies Collaboration (2002) Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA 288:2015–2022

    Article  Google Scholar 

  2. Castañon MM, Lauricella AM, Kordich L, Quintana I (2007) Plasma homocysteine cutoff values for venous thrombosis. Clin Chem Lab Med 45:232–236

    Article  Google Scholar 

  3. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 346:476–483

    Article  CAS  Google Scholar 

  4. Mudd SH, Finkelstein JD, Refsum H, Ueland PM, Malinow MR, Lentz SR, Jacobsen DW, Brattström L, Wilcken B, Wilcken DE, Blom HJ, Stabler SP, Allen RH, Selhub J, Rosenberg IH (2000) Homocysteine and its disulfide derivatives: a suggested consensus terminology. Arterioscler Thromb Vasc Biol 20:1704–1706

    Article  CAS  Google Scholar 

  5. Jakubowski H, Goldman E (1993) Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells. FEBS Lett 317:237–240

    Article  CAS  Google Scholar 

  6. Blom HJ (2000) Consequences of homocysteine export and oxidation in the vascular system. Semin Thromb Hemost 26:227–232

    Article  CAS  Google Scholar 

  7. Jakubowski H (1999) Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J 13:2277–2283

    CAS  Google Scholar 

  8. Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316

    Article  CAS  Google Scholar 

  9. Radi R (2004) Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci USA 101:4003–4008

    Article  CAS  Google Scholar 

  10. Vlassara H (1994) Recent progress on the biologic and clinical significance of advanced glycosylation end products. J Lab Clin Med 124:19–30

    CAS  Google Scholar 

  11. Jakubowski H (2004) Molecular basis of homocysteine toxicity in humans. Cell Mol Life Sci 61:470–487

    Article  CAS  Google Scholar 

  12. Jalili S, Yousefi R, Papari MM, Moosavi-Movahedi AA (2011) Effect of homocysteine thiolactone on structure and aggregation propensity of bovine pancreatic insulin. Protein J 30:299–307

    Article  CAS  Google Scholar 

  13. Perła-Kaján J, Marczak Ł, Kaján L, Skowronek P, Twardowski T, Jakubowski H (2007) Modification by homocysteine thiolactone affects redox status of cytochrome C. Biochemistry 29:6225–6231

    Article  Google Scholar 

  14. Jakubowski H (2002) Homocysteine is a protein amino acid in humans: implications for homocysteine-linked disease. J Biol Chem 23:30425–30428

    Article  Google Scholar 

  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  16. Clarke HG, Freeman T (1968) Quantitative immunoelectrophoresis of human serum proteins. Clin Sci 35:403–413

    CAS  Google Scholar 

  17. Ray JG (1998) Meta-analysis of hyperhomocysteinemia as a risk factor for venous thromboembolic disease. Arch Inter Med 158:2101–2116

    Article  CAS  Google Scholar 

  18. Vollset SE, Refsum H, Tverdal A et al (2001) Plasma total homocysteine and cardiovascular and noncardiovascular mortality: the Hordaland Homocysteine Study. Am J Clin Nutr 74:130–136

    CAS  Google Scholar 

  19. Fallon UB, Ben-Shlomo Y, Elwood P, Ubbink JB, Smith GD (2001) Homocysteine and coronary heart disease in the Caerphilly cohort: a 10 year follow up. Heart 85:153–158

    Article  CAS  Google Scholar 

  20. Bønaa KH, Njølstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, Wang H, Nordrehaug JE, Arnesen E, Rasmussen K, NORVIT Trial Investigators (2006) Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 354:1578–1588

    Article  Google Scholar 

  21. Spence JD (2013) B vitamin therapy for homocysteine: renal function and vitamin B12 determine cardiovascular outcomes. Clin Chem Lab Med 51:633–637

    Article  CAS  Google Scholar 

  22. Jakubowski H (2007) The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin Chem Lab Med 45:1704–1716

    Article  CAS  Google Scholar 

  23. Paoli P, Sbrana F, Tiribilli B, Caselli A, Pantera B, Cirri P, De Donatis A, Formigli L, Nosi D, Manao G, Camici G, Ramponi G (2010) Protein N-homocysteinylation induces the formation of toxic amyloid-like protofibrils. J Mol Biol 400:889–907

    Article  CAS  Google Scholar 

  24. Gates AT, Lowry M, Fletcher KA, Murugeshu A, Rusin O, Robinson JW, Strongin RM, Warner IM (2007) Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization. Anal Chem 79:8249–8256

    Article  CAS  Google Scholar 

  25. Chwatko G, Jakubowski H (2005) The determination of homocysteine-thiolactone in human plasma. Anal Biochem 337:271–277

    Article  CAS  Google Scholar 

  26. Marczak L, Sikora M, Stobiecki M, Jakubowski H (2011) Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry. J Proteomics 74:967–974

    Article  CAS  Google Scholar 

  27. Glowacki R, Jakubowski H (2004) Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation. J Biol Chem 279:10864–10871

    Article  CAS  Google Scholar 

  28. Perna AF, Satta E, Acanfora F, Lombardi C, Ingrosso D, De Santo NG (2006) Increased plasma protein homocysteinylation in hemodialysis patients. Kidney Int 69:869–876

    Article  CAS  Google Scholar 

  29. Yang X, Gao Y, Zhou J, Zhen Y, Yang Y, Wang J, Song L, Liu Y, Xu H, Chen Z, Hui R (2006) Plasma homocysteine thiolactone adducts associated with risk of coronary heart disease. Clin Chim Acta 364:230–234

    Article  CAS  Google Scholar 

  30. Undas A, Perła J, Lacinski M, Trzeciak W, Kaźmierski R, Jakubowski H (2004) Autoantibodies against N-homocysteinylated proteins in humans: implications for atherosclerosis. Stroke 35:1299–1304

    Article  CAS  Google Scholar 

  31. Undas A, Jankowski M, Twardowska M, Padjas A, Jakubowski H, Szczeklik A (2005) Antibodies to N-homocysteinylated albumin as a marker for early-onset coronary artery disease in men. Thromb Haemost 93:346–350

    CAS  Google Scholar 

  32. Jakubowski H, Zhang L, Bardeguez A, Aviv A (2000) Homocysteine thiolactone and protein homocysteinylation in human endothelial cells: implications for atherosclerosis. Circ Res 87:45–51

    Article  CAS  Google Scholar 

  33. Capasso R, Sambri I, Cimmino A, Salemme S, Lombardi C, Acanfora F, Satta E, Puppione DL, Perna AF, Ingrosso D (2012) Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesión and up-regulation of MCP1, Hsp60 and ADAM17. PLoS ONE 7(2):e31388. doi:10.1371/journal.pone.0031388

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by a Grant from the University of Buenos Aires (UBACYT 01/W 998), Argentina.

Conflict of interest

All authors disclose no financial or personal relationships with other people or organizations that could inappropriately influence this work.

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Authors and Affiliations

  1. Laboratory of Hemostasis and Thrombosis, Department of Biological Chemistry, School of Exact and Natural Sciences, University of Buenos Aires, Pabellón II, piso 4°, Ciudad Universitaria, Int. Güiraldes 2160 (C1428EHA), Buenos Aires, Argentina

    Valeria Genoud, Mercedes Castañon, Ana María Lauricella & Irene Quintana

Authors
  1. Valeria Genoud
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  2. Mercedes Castañon
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  3. Ana María Lauricella
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  4. Irene Quintana
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Corresponding author

Correspondence to Irene Quintana.

Additional information

In memory of Mercedes Castañon.

Valeria Genoud and Mercedes Castañon have contributed equally to this work.

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Genoud, V., Castañon, M., Lauricella, A.M. et al. Characterization of N-homocysteinylated Albumin Adducts. Protein J 33, 85–91 (2014). https://doi.org/10.1007/s10930-013-9540-z

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