Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcythiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.

A protocol for the identification of N-homocysteinylation sites in plasma proteins is described. Human plasma or purified fibrinogen is subjected to trypsin digestion and analysis of N-Hcy-peptides by liquid chromatography/mass spectroscopy (LC/MS). Human fibrinogen is isolated from the plasma by the glycine precipitation method. Identification of N-Hcy-Lys-peptides in tryptic digests of in vivo-derived samples is facilitated by the use of N-Hcy-albumin and N-Hcy-fibrinogen synthesized in vitro from commercially available human proteins. This protocol allows identification of N-homocysteinylation sites at Lys4, Lys12, Lys137, and Lys525 in albumin directly in trypsin-digested human serum samples. N-Hcy-Lys562, N-Hcy-Lys344, and N-Hcy-Lys385 were identified in human fibrinogen from patients with cystathionine β-synthase deficiency. The protocol can be completed in 4 days.

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