N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 82

receptor sites or from directly binding to the receptor [334]. N-Hcy-LDL
containing from 5 to 50 N-Hcy residues per protein molecule can be prepared
by incubation with Hcy-thiolactone [335]. At modification level of 9 N-Hcy

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residues per LDL molecule ( 2.5 % lysine residues modified), some aggregation

occurs, but is reversed by DTT, suggesting that it is caused by disulfide bond
formation. At 8 N-Hcy residues per LDL molecule, N-Hcy-LDL exhibited the
same mobility on paper electrophoresis, gel filtration elution pattern, and particle
size (20 nm) as native LDL. At this extent of modification, the affinity of N-
Hcy-LDL for LDL receptors and subsequent internalization by L 2 C lymphocytes
is identical to that of native LDL.
Higher extent of N-homocysteinylation causes increase in density, faster elec-
trophoretic mobility, and aggregation and precipitation of N-Hcy-LDL [336]. Such
highly modified N-Hcy-LDL is taken up and degraded faster than native LDL and
causes increased cholesterol accumulation in human macrophages. Degradation of
N-Hcy-LDL and native LDL by macrophages is inhibited 75.6 % and 11.4 %,
respectively, by cytochalasin B, suggesting increased uptake of aggregated N-Hcy-
LDL by phagocytosis. Highly modified N-Hcy-LDL, containing about 90 N-Hcy
residues per LDL molecule (25 % lysine residues modified), exhibits decreased
binding, internalization, and degradation by normal human fibroblasts.
A more recent study shows that N-Hcy-LDL containing about 25 N-Hcy residues
per LDL molecule has fluorescence characteristics and levels of lipid
hydroperoxides similar to those of native LDL, suggesting that N-homocystei-
nylation does not significantly affect its structure and does not cause oxidative
damage [170]. Treatment with N-Hcy-LDL, but not with native LDL, induces
significant increase in the levels of hydroperoxides in human aortic endothelial
cells, suggesting that N-Hcy-LDL induces oxidative damage in these cells. At the
same time, a significant decrease in cell viability in N-Hcy-LDL-treated cells, but
not in native LDL-treated cells, is observed [170]. Furthermore, incubation of
endothelial cells with N-Hcy-LDL causes a significant increase in cytoplasmic
calcium levels and peroxynitrite production and a decrease in Na + /K + -ATPase and
nitric oxide production in these cells, compared with cell incubated with control
LDL. In addition, a positive correlation is observed between Na + /K + -ATPase activ-
ity and cytoplasmic Ca 2+ content and between peroxynitrite activity and cytoplas-
mic Ca 2+ content. These findings show that N-Hcy-LDL induces alterations in
functional properties and nitric acid metabolism of human endothelial cells [337].

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5.4.4 N -Homocysteinylation and HDL Function

High-density lipoprotein plays a central role in reverse cholesterol transport and has
anti-inflammatory and antioxidant properties. These activities are responsible for
atheroprotective roles of HDL. In addition to lipids, HDL contains ApoA1 as a major
protein component and Hcy-thiolactonase/PON1 as a minor protein component.
Human HDL carries small amounts of N-linked Hcy (Tables 5.4 and 5.5 ) [79, 303]
and is susceptible to N-homocysteinylation by Hcy-thiolactone in vitro [320].