N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 59

5.2 Biological Formation

Biological formation of N-Hcy-protein has originally been discovered in tissue
cultures of human cells (Fig. 3.7 ) [73, 74] and subsequently demonstrated to occur
in humans [115] and mice [113]. The biological mechanism involves two steps. In
the first step, Hcy is metabolically converted to Hcy-thiolactone by methionyl-
tRNA synthetase (Reactions 3.7 and 3.8 ). In the second step, Hcy-thiolactone reacts
with protein lysine residues, affording N-Hcy-protein (Reaction 3.4 ). These
reactions have also been demonstrated in in vitro studies with purified components.
Evidence supporting this mechanism includes precursor-product relationships
between Hcy, Hcy-thiolactone, and N-Hcy-protein, observed both in ex vivo tissue
culture studies and in vivo in humans and mice. The observations that mutations
in genes encoding enzymes that participate in Hcy metabolism lead to increases in
N-Hcy-protein levels add further support for this mechanism. Additional supporting
evidence comes from the identification of specific N-Hcy-lysine residues in human
serum albumin [96, 212, 213] and fibrinogen [215] in vivo.

5.2.1 N -Hcy-Protein Levels In Vivo

The demonstration that protein N-homocysteinylation occurs in intact organisms
in vivo came with the discovery of N-linked Hcy in human plasma proteins, first
reported in 2000 [139]. Subsequent studies have confirmed this finding and
established that normal human plasma contains 0.49 0.08 μ M [79, 115, 220],

0.51 M [301] protein N-linked Hcy. Several

genetic and dietary factors that affect the levels of N-Hcy-protein in humans and
mice have been identified. In humans, these include mutations in the CBS and
MTHFR genes, polymorphic variations in the PON1 gene. In mice, known
determinants of N-Hcy-protein include Cbs, Mthfr, Pcft, Pon1, and Blmh genes,
as well as the diet.
Protein N-linked Hcy increases significantly in hyperhomocysteinemia. For
example, in CBS- and MTHFR-deficient patients, plasma protein N-linked Hcy
levels are elevated up to 31.4-fold (Table 3.12 ). Furthermore, CBS-deficient
patients have also up to ninefold higher plasma levels of prothrombotic N-Hcy-
fibrinogen than normal subjects [115].
Genetic or nutritional disorders in Hcy or folate metabolism increase protein N-
homocysteinylation also in experimental animals [113]. For example, the inactiva-
tion of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene causes
10-30-fold increase in plasma N-Hcy-protein levels (Tables 5.1 and 5.2 ). Liver
N-Hcy-protein is elevated 3.4-fold in severely hyperhomocysteinemic Cbs-
deficient mice, 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice,
and 3.6-fold in severely hyperhomocysteinemic Pcft-null mice. However, liver N-
Hcy-protein levels are similar in Mthfr-null mice (49.8

0.11 μ M [300], and 0.35 013 μ

38.2 pmol/mg protein)