N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 66

Table 5.9 N-linked Hcy and S-linked Hcy levels in mouse pelage keratin [308]
Genotype
(n)

N-Hcy, pmol/mg
hair

S-Hcy, pmol/mg
hair

N-Hcy/(N-Hcy + S-
Hcy)

SDS-soluble N-Hcy
fraction

Wild type
(12)

87 10 127 10 0.59 0.05 0.25 0.05

Cbs / (9)

1,056 104 2,138 360 0.33 0.03 0.04 0.00

Cse / (5) 546 41 682 129 0.55 0.03 0.14 0.01

Mthfr /
(4)

237 23 356 74 0.40 0.06 0.21 0.03

which only 4 % is SDS soluble, compared with 25 % SDS-soluble N-Hcy-keratin in
wild-type animals. These findings suggest that N-homocysteinylation causes kera-
tin damage.
In vitro studies have shown that N-homocysteinylation causes protein damage
(Fig. 5.11 ). The findings that increased keratin N-homocysteinylation in
hyperhomocysteinemic mice decreases its solubility provide the first evidence
that protein damage induced by N-homocysteinylation occurs in vivo. The defect
in keratin solubility associated with N-homocysteinylation [308] can explain pelage
abnormalities observed in Cbs / mice [309].

5.2.2 Site-Specific N -Homocysteinylation In Vivo

Identification of specific N-Hcy-Lys residues in proteins in vivo provides direct
support for a conclusion that protein N-homocysteinylation in humans occurs by a
mechanism involving the reaction of Hcy-thiolactone with protein lysine residues
(Reaction 3.4 ). So far, site-specific N-homocysteinylation in vivo has been analyzed
for human serum albumin [212, 213], fibrinogen [215], and dynein [299].
Three albumin residues, Lys525, 137, and 212, are found to be N-
homocysteinylated in vivo in human plasma from CBS-deficient patients and
unaffected individuals, with Lys525 being the predominant in vivo-modified site
(Fig. 5.2 ). Albumin peptide containing N-Hcy-Lys525 is identified in essentially all
analyzed plasma samples (43 out of 44), including those that had the lowest tHcy
concentration (9.9 μM), whereas peptides containing N-Hcy-Lys137 and N-Hcy-
Lys212 are identified in albumin from CBS-deficient patients whose plasma tHcy
concentration was elevated, at least 34.9

11.0 μ M and 131 21 μ M, respec-

tively [212].
Three lysine residues carry N-linked Hcy in fibrinogen isolated from CBS-
deficient patients, one in each subunit: Lys562 in

α -subunit, Lys344 in β -subunit,

and Lys385 in -subunit (Fig. 5.3 ). These three in vivo N-homocysteinylation sites

are also predominant sites for fibrinogen N-homocysteinylation in vitro [215].

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