Discoveries of Protein S- and N-Homocysteinylation - Homocysteine in Protein Structure/Function and Human Disease - page 53

Chapter 4
Discoveries of Protein S- and
N-Homocysteinylation

In early studies, Hcy was identified in plasma and urine from patients with CBS or
MTHFR deficiency [290, 291], but was undetectable in normal individuals. What
was surprising in those studies was the inability to detect Hcy in tissues from CBS-
or MTHFR-deficient patients [292, 293]. This suggested that a significant quantity
of Hcy must have escaped detection by the conventional methods of amino acid
analysis [294], possibly because Hcy was bound to protein via disulfide bonds and
removed during the deproteinization step.
Indeed, a simple treatment with the disulfide-reducing agent 2-mercapto-
ethanol led to the discovery of Hcy in normal individuals and revealed that
most plasma and tissue Hcy is linked to protein via disulfide bonds [91]. It
was also demonstrated that exogenous Hcy added to plasma quickly becomes
protein bound and can be quantitatively released by the 2-mercaptoethanol
treatment. These findings provided the first evidence for a redox reaction
of Hcy with plasma proteins that is now called protein S-homocysteinylation
[41, 295]. Subsequent studies have identified albumin [103] and IgG [79] as
the major carriers of S-linked Hcy in plasma. Albumin also carries most of
plasma Cys [111], which forms a disulfide bond with the conserved Cys34
thiol [108, 296].
Protein N-homocysteinylation was first discovered in studies of Hcy-thiolactone
metabolism in cultured human cells using [ 35 S]Met and [ 35 S]Hcy as tracers. Those
studies demonstrated that fibroblasts from CBS-deficient patients and the
oncogenically transformed cells from breast cancer patients produce more Hcy-
thiolactone and N-Hcy-protein than normal cells from unaffected individuals [73].
The treatment of cells with the antifolate drug aminopterin increases N-Hcy-
protein levels, in addition to increasing Hcy-thiolactone and Hcy. In human
endothelial cells,

the extent of protein N-homocysteinylation increases with