S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 121

onset of senile systemic amyloidosis [418]. It is likely that S-homocysteinylation
similarly increases amyloidogenicity of TTR; this, however, remains to be

demonstrated.
It has been stated that N-Hcy-transthyretin is undetectable in human plasma
[104]. However, the authors acknowledge that their inability to detect N-Hcy-
transthyretin is due to the interference from a signal corresponding to S-Cys-
transthyretin and to low sensitivity of their assay: the detection limit of the
LC-MS assay used by Sass et al. [104] is 1 % relative to total transthyretin. This
limit of detection is several orders of magnitude less sensitive than that of the HPLC
with fluorescence detection method, which allows quantification of as little as
0.00006 mol N-linked Hcy/mol protein (or 0.006 mol%) [297].

7.1.3 Apolipoprotein B and Lipoprotein[a]

ApoB-100, the major protein of LDL, is composed of a single polypeptide chain of
4,563 amino acid residues and contains a site for binding to the cell-surface LDL
receptor. Sixteen of the 25 cysteine residues exist in the disulfide form, while nine
cysteine residues have free sulfhydryl groups [419]. Each of the low molecular
weight thiols present in plasma has been reported to bind to ApoB via disulfide
bonds. Thus, ApoB-100 isolated from normal human plasma carries 0.0137 mol S-
linked Hcy/mol ApoB [420], and the content of S-linked Cys, CysGly, GSH, and
γ GluCys (in mol/mol ApoB-100) is 0.324, 0.108, 0.0095, and 0.0026, respectively.

Plasma tHcy is a major determinant of S-Hcy-ApoB-100 levels.
Incubation of ApoB-100 with increasing concentrations of Hcy (up to 0.1 mM)
leads to increased binding with saturation at about 0.2 mol S-linked Hcy/mol ApoB
[144]. Increase of Hcy concentration to 5 mM does not lead to any further increase
in Hcy binding. This means that Hcy is able to form disulfide bonds with only a very
small fraction (2.2 %) of the 9 cysteine thiols that are present in ApoB-100. The
reasons for such an inefficient binding are not clear but raise a possibility that a
minor variant of ApoB or a contaminating protein, and not ApoB-100 itself, is
responsible for Hcy binding.
The treatment of cultured human endothelial cells with S-Hcy-ApoB-100
prepared in vitro led to increased generation of reactive oxygen species, decreased
proliferation, and increased cytotoxicity [144]. Whether these effects are specific to
S-thiolation of ApoB-100 by Hcy and whether S-thiolation by other thiols, in
particular the much more abundant thiolation with cysteine, affects interactions of
ApoB-100 with endothelial cells have not been examined.
S-Hcy-ApoB-100 is reported to be significantly elevated (to 0.024 mol S-linked
Hcy/mol ApoB) in chronic kidney disease (CKD) patients in comparison with
healthy controls [421]. S-Cys-ApoB-100 is also significantly elevated in these
patients to 0.386 mol S-linked Hcy/mol ApoB. In CKD patients, plasma S-Hcy-
ApoB-100 varies from 0.01 to 0.06 mol S-Hcy/mol ApoB while tHcy varies from
8to60

μ M. Plasma S-Hcy-ApoB is positively correlated with tHcy and creatinine.