N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 68

a b c

600

80

4

Blmh-/-
WT

Blmh-/-
WT

70

500

Blmh-/-
WT

3

60

400

50

300

2

40

30

200

1

20

100

10

0 0

0

0 20 40 60 80 100 0 12 24 36 48 60

0 5 10 15 20 25 30

Time, min Time, h

Time, min

Fig. 5.4 Kinetics of plasma Hcy-thiolactone (a), total Hcy (b), and N-Hcy-protein (c) turnover in
mice. For Hcy-thiolactone (a) and total Hcy (b) turnover experiments, mice have been injected i.p.
with 600 nmol L -Hcy-thiolactone/g body weight. For N-Hcy-protein (c) turnover experiments,
2,850 nmol L -Hcy-thiolactone/g body weight L -Hcy-thiolactone was used. Metabolites were
analyzed at indicated times post-injection and data points were fitted to an exponential equation
[A t ] [A 0 ]·e k·t , where k is a first-order rate constant, [A t ] is metabolite concentration measured

at time t, and [A 0 ] is metabolite concentration extrapolated to time zero. Representative kinetics
obtained for individual knockout Blmh / (filled circle) and wild-type Blmh +/+

¼

(multiplication

sign) mice are shown (Reproduced from [141])

5.3 Turnover

An indirect evidence for the proteolytic degradation of N-Hcy-protein is provided by
the discovery of anti-N-Hcy-protein IgG autoantibodies [172, 310], which specifi-
cally recognize Nε -Hcy-Lys epitopes and whose formation can only be initiated by

proteolytic degradation of N-Hcy-protein to antigenic peptides that are then displayed
on the cell surface. First direct evidence for N-Hcy-protein turnover came frommouse
studies. Wild-type mice fed with a high-Met diet accumulate up to 22 M N-Hcy-

protein in plasma [113]. These mice have also elevated plasma Hcy-thiolactone (from
6to80nM)andtHcylevels(from3.5to126

μ

M). Shifting the mice from high-Met
to normal chow diet normalizes Hcy-thiolactone and tHcy levels and lowers
N-Hcy-protein to 3.6

μ

M, indicating that N-Hcy-proteins are turned over. Direct
kinetic measurements indicate that N-Hcy-protein turns over with a half-life of
10.2

μ

1.4 h in the mouse plasma [141] (Fig. 5.4 ). The clearance of plasma N-Hcy-
protein is 24-fold slower than the clearance of plasma Hcy (half-life of 26.2 min)
and 120-fold slower than the clearance of plasma Hcy-thiolactone (half-life of
5.1 min) (Fig. 5.4 ) [140, 141]. N-Hcy-protein is turned over by proteolytic degradation
with the liberation of the isopeptide Nε

-Hcy-Lys (Fig. 5.5 )[72].

5.3.1 N

ε -Homocysteinyl-Lysine

5.3.1.1 Chemical Synthesis

Nε -Hcy-Lys isopeptide has been originally identified in vitro on TLC plates as a

product of facile reaction of Hcy-thiolactone with lysine [73, 139]. This reaction