N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 83

Under physiological conditions in vivo, one or two lysine residues, out of the 21
lysine residues present in ApoA1 are N-homocysteinylated [303]. In vitro, however,
5 or 6 lysine residues can be N-homocysteinylated using high concentrations of
Hcy-thiolactone. The susceptibility of HDL to N-homocysteinylation by Hcy-
thiolactone in vitro is negatively correlated with the paraoxonase activity [320]
(which reflects the native Hcy-thiolactonase activity of PON1). Similar protection
against N-homocysteinylation by high activity of Hcy-thiolactonase/PON1 is
observed for total serum protein not only in vitro but also in vivo in humans
[152, 250].
N-Homocysteinylation causes an increase in the intensity and a blue shift of the
intrinsic Trp fluorescence of HDL [320]. Similar changes are observed in the
external fluorescent lipophilic probe Lourdan that binds at the lipid-water interface
of HDL. This indicates that N-homocysteinylation induces structural changes in the
environment of Trp residues as well as in the Lourdan binding site of HDL [320].
N-Hcy HDL has a significantly decreased paraoxonase activity relative to native
untreated HDL. These results indicate that the modification by Hcy-thiolactone
affects structure and function of HDL.

5.4.5 N -Homocysteinylation Induces Protein Aggregation

and Amyloidal Conversion

Early studies of protein modification by Hcy-thiolactone have shown that N-Hcy-
proteins have a tendency to aggregate and precipitate from solutions [78]. Some
proteins, such as RNaseA, trypsin, and cytochrome c, aggregate and precipitate
when the extent of N-homocysteinylation is low, 1-2 mol N-Hcy/mol protein
(Fig. 5.11 ). Other proteins, such as fibrinogen, albumin, IgG, and transferrin, are
fully soluble even when the extent of N-homocysteinylation is as high as 20 mol N-
Hcy/mol protein but eventually begin to precipitate when the extent of modification
is higher. Other proteins, such as hemoglobin and myoglobin, exhibited intermedi-
ate sensitivity to N-homocysteinylation (Fig. 5.11 ). Subsequent studies have shown
that protein aggregation elicited by N-homocysteinylation involves amyloid
conversion even in proteins with mostly helical structures, such as albumin.

5.4.5.1 N -Hcy-Albumin

Serum albumin is an inherently stable globular protein with mostly -helical

structure. Incubation of albumin in diluted solutions for several weeks at 37 C
does not lead to protein aggregation. However, incubation over extended periods of
time (7 days or longer) of serum albumin modified with Hcy-thiolactone confers on
the protein the ability to bind thioflavin T which induces fluorescence (a positive
test for the presence of

α

β -sheet structures characteristic of amyloids). This reveals