Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Metabolism and neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin hydrolase


Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze l-Hcy-thiolactone (but not d-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh −/− mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh +/+ animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh −/− mice relative to Blmh +/+ animals. Plasma N-Hcy-protein was elevated in Blmh −/− mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh +/+ animals. More intraperitoneally injected l-Hcy-thiolactone was recovered in plasma in Blmh −/− mice than in wild-type Blmh +/+ animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh +/+ mice injected intraperitoneally with d-Hcy-thiolactone, d,l-Hcy-thiolactone, or l-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by l-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh −/− than in Blmh +/+ mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against l-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer’s disease.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2



Bleomycin hydrolase


Cystathionine β-synthase




Total Hcy




Methylenetetrahydrofolate reductase


  1. Bromme D, Rossi AB, Smeekens SP, Anderson DC, Payan DG (1996) Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization. Biochemistry 35:6706–6714

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

  3. Chwatko G, Jakubowski H (2005b) Urinary excretion of homocysteine-thiolactone in humans. Clin Chem 51:408–415

  4. Chwatko G, Boers GH, Strauss KA, Shih DM, Jakubowski H (2007) Mutations in methylenetetrahydrofolate reductase or cystathionine beta-synthase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice. FASEB J 21:1707–1713

  5. Endo N, Nishiyama K, Otsuka A, Kanouchi H, Taga M, Oka T (2006) Antioxidant activity of vitamin B6 delays homocysteine-induced atherosclerosis in rats. Br J Nutr 95:1088–1093

  6. Folbergrova J (1997) Anticonvulsant action of both NMDA and non-NMDA receptor antagonists against seizures induced by homocysteine in immature rats. Exp Neurol 145:442–450

  7. 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

  8. Glowacki R, Bald E, Jakubowski H (2010) Identification and origin of Nε-homocysteinyl-lysine isopeptide in humans and mice. Amino Acids 39:1563–1569

  9. Gu W, Lu J, Yang G, Dou J, Mu Y, Meng J, Pan C (2008) Plasma homocysteine thiolactone associated with risk of macrovasculopathy in Chinese patients with type 2 diabetes mellitus. Adv Ther 25:914–924

  10. Harker LA, Slichter SJ, Scott CR, Ross R (1974) Homocystinemia. Vascular injury and arterial thrombosis. N Engl J Med 291:537–543

  11. Jakubowski H (1990) Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli. Proc Natl Acad Sci USA 87:4504–4508

  12. Jakubowski H (1997) Metabolism of homocysteine thiolactone in human cell cultures. Possible mechanism for pathological consequences of elevated homocysteine levels. J Biol Chem 272:1935–1942

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

  14. Jakubowski H (2000a) Homocysteine thiolactone: metabolic origin and protein homocysteinylation in humans. J Nutr 130:377S–381S

  15. Jakubowski H (2000b) Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N-homocysteinylation. J Biol Chem 275:3957–3962

  16. Jakubowski H (2002) Homocysteine is a protein amino acid in humans. Implications for homocysteine-linked disease. J Biol Chem 277:30425–30428

  17. Jakubowski H (2006) Mechanism of the condensation of homocysteine thiolactone with aldehydes. Chemistry 12:8039–8043

  18. Jakubowski H (2007) Facile syntheses of [35S]homocysteine-thiolactone, [35S]homocystine, [35S]homocysteine, and [S-nitroso-35S]homocysteine. Anal Biochem 370:124–126

  19. Jakubowski H (2008a) The pathophysiological hypothesis of homocysteine thiolactone-mediated vascular disease. J Physiol Pharmacol 59(Suppl 9):155–167

  20. Jakubowski H (2008b) New method for the determination of protein N-linked homocysteine. Anal Biochem 380:257–261

  21. Jakubowski H (2011) Quality control in tRNA charging—editing of homocysteine. Acta Biochim Pol 58:149–163

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

  23. 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

  24. Jakubowski H, Boers GH, Strauss KA (2008) Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans. FASEB J 22:4071–4076

  25. Jakubowski H, Perla-Kajan J, Finnell RH, Cabrera RM, Wang H, Gupta S, Kruger WD, Kraus JP, Shih DM (2009) Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice. FASEB J 23:1721–1727

  26. Kajiya A, Kaji H, Isobe T, Takeda A (2006) Processing of amyloid beta-peptides by neutral cysteine protease bleomycin hydrolase. Protein Pept Lett 13:119–123

  27. Kamata Y, Itoh Y, Kajiya A, Karasawa S, Sakatani C, Takekoshi S, Osamura RY, Takeda A (2007) Quantification of neutral cysteine protease bleomycin hydrolase and its localization in rat tissues. J Biochem 141:69–76

  28. Kamata Y, Taniguchi A, Yamamoto M, Nomura J, Ishihara K, Takahara H, Hibino T, Takeda A (2009) Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids. J Biol Chem 284:12829–12836

  29. Lefterov IM, Koldamova RP, Lefterova MI, Schwartz DR, Lazo JS (2001) Cysteine 73 in bleomycin hydrolase is critical for amyloid precursor protein processing. Biochem Biophys Res Commun 283:994–999

  30. Mudd SH, Levy HL, Krauss JP (2001) Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B (eds) The metabolic and molecular bases of inherited disease, vol 2. Mc Graw-Hill, New York, pp 2007–2056

  31. 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

  32. Papassotiropoulos A, Bagli M, Jessen F, Frahnert C, Rao ML, Maier W, Heun R (2000) Confirmation of the association between bleomycin hydrolase genotype and Alzheimer’s disease. Mol Psychiatry 5:213–215

  33. Perla-Kajan J, Jakubowski H (2010) Paraoxonase 1 protects against protein N-homocysteinylation in humans. FASEB J 24:931–936

  34. Perla-Kajan J, Marczak L, Kajan L, Skowronek P, Twardowski T, Jakubowski H (2007) Modification by homocysteine thiolactone affects redox status of cytochrome C. Biochemistry 46:6225–6231

  35. Perla-Kajan J, Stanger O, Luczak M, Ziolkowska A, Malendowicz LK, Twardowski T, Lhotak S, Austin RC, Jakubowski H (2008) Immunohistochemical detection of N-homocysteinylated proteins in humans and mice. Biomed Pharmacother 62:473–479

  36. Rasic-Markovic A, Stanojlovic O, Hrncic D, Krstic D, Colovic M, Susic V, Radosavljevic T, Djuric D (2009) The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration. Mol Cell Biochem 327:39–45

  37. Sauls DL, Lockhart E, Warren ME, Lenkowski A, Wilhelm SE, Hoffman M (2006) Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia. Biochemistry 45:2480–2487

  38. Schwartz DR, Homanics GE, Hoyt DG, Klein E, Abernethy J, Lazo JS (1999) The neutral cysteine protease bleomycin hydrolase is essential for epidermal integrity and bleomycin resistance. Proc Natl Acad Sci USA 96:4680–4685

  39. Selhub J, Troen A, Rosenberg IH (2010) B vitamins and the aging brain. Nutr Rev 68(Suppl 2):S112–S118

  40. Sibrian-Vazquez M, Escobedo JO, Lim S, Samoei GK, Strongin RM (2010) Homocystamides promote free-radical and oxidative damage to proteins. Proc Natl Acad Sci USA 107:551–554

  41. Smith AD (2008) The worldwide challenge of the dementias: a role for B vitamins and homocysteine? Food Nutr Bull 29:S143–S172

  42. Smith AD, Smith SM, de Jager CA, Whitbread P, Johnston C, Agacinski G, Oulhaj A, Bradley KM, Jacoby R, Refsum H (2010) Homocysteine-lowering by B vitamins slows the rate of accelerated brain atrophy in mild cognitive impairment: a randomized controlled trial. PLoS One 5:e12244

  43. Spence AM, Rasey JS, Dwyer-Hansen L, Grunbaum Z, Livesey J, Chin L, Nelson N, Stein D, Krohn KA, Ali-Osman F (1995) Toxicity, biodistribution and radioprotective capacity of l-homocysteine thiolactone in CNS tissues and tumors in rodents: comparison with prior results with phosphorothioates. Radiother Oncol 35:216–226

  44. Sprince H, Parker CM, Josephs JA Jr, Magazino J (1969) Convulsant activity of homocysteine and other short-chain mercaptoacids: protection therefrom. Ann N Y Acad Sci 166:323–325

  45. Suszynska J, Tisonczyk J, Lee HG, Smith MA, Jakubowski H (2010) Reduced homocysteine-thiolactonase activity in Alzheimer’s disease. J Alzheimers Dis 19:1177–1183

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

  47. 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

  48. Velez-Carrasco W, Merkel M, Twiss CO, Smith JD (2008) Dietary methionine effects on plasma homocysteine and HDL metabolism in mice. J Nutr Biochem 19:362–370

  49. 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

  50. Zabczyk M, Glowacki R, Machnik A, Herod P, Kazek G, Jakubowski H, Undas A (2011) Elevated concentrations of Nε-homocysteinyl-lysine isopeptide in acute myocardial infarction: links with ADMA formation. Clin Chem Lab Med 49:729–735

  51. Zhou J, Moller J, Danielsen CC, Bentzon J, Ravn HB, Austin RC, Falk E (2001) Dietary supplementation with methionine and homocysteine promotes early atherosclerosis but not plaque rupture in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 21:1470–1476

  52. Zimny J, Sikora M, Guranowski A, Jakubowski H (2006) Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase. J Biol Chem 281:22485–22492

Download references


We thank John Lazo for kindly providing Blmh null mice and for his comments on the manuscript. This work was supported in part by grants from the American Heart Association (0855919D) and the National Science Center, Poland (DEC-2011/01/B/NZ1/03417).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Correspondence to Hieronim Jakubowski.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Borowczyk, K., Tisończyk, J. & Jakubowski, H. Metabolism and neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin hydrolase. Amino Acids 43, 1339–1348 (2012). https://doi.org/10.1007/s00726-011-1207-5

Download citation


  • Bleomycin hydrolase
  • Homocysteine thiolactone
  • Brain
  • Seizures
  • Alzheimer’s disease