S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 123

content of 13.4 % cysteine residues, fibrillin-1 is one of the most-cysteine-rich
proteins. Cysteine residues form intradomain disulfide bonds important for the
correct folding and structure of each domain. Disruption of the connective tissue
structure contributes to the pathogenic mechanisms underlying two inherited
diseases with very different etiologies: Marfan syndrome and homocystinuria.
Marfan syndrome is a connective tissue disease caused by mutations in the
fibrillin-1 gene. Many missense mutations cause fibrillin-1 domain misfolding,
which may affect the delivery of fibrillin-1 and/or its assembly into microfibrils.
Patients with homocystinuria often exhibit skeletal abnormalities resembling the
Marfan syndrome phenotype [20, 46], suggesting that elevated Hcy levels may lead
to chemical reduction of disulfide bonds within fibrillin-1 domains resulting in the
loss of native structure.
In vitro experiments show that Hcy reduces disulfide bonds of recombinant
fibrillin-1 cbEGF domain fragments, which results in structural changes that lead
to misfolding of the protein [432]. Mass spectroscopic analyses identify Hcy
residues attached via disulfide bonds to fibrillin-1 fragments. Calcium binding
protects domain structure of the protein against reduction by Hcy. Circular dichro-
ism spectroscopy reveals moderate changes in the secondary structure of recombi-
nant fragments spanning the entire human fibrillin-1 molecule after treatment with
Hcy [433]. S-homocysteinylation affects also functional properties of the fibrillin-1
fragments. For example, calcium binding to S-homocysteinylated fragments is
completely abolished, and the fragments containing S-linked Hcy become signifi-
cantly more susceptible to proteolytic degradation [433].
S-homocysteinylated fibrillin-1 fragments exhibit abnormal self-interaction,
reduced multimerization of the C terminus, and impaired coacervation properties
[434]. The deposition of the fibrillin-1 network by human dermal fibroblasts is
reduced by the treatment of the cells with 1-mM Hcy, but not with 0.3-mM Hcy.
These effects are specific to S-homocysteinylation and are not observed after
S-cysteinylation. However, binding of fibrillin-1 to heparin through several domains
distributed throughout the molecule is inhibited similarly by Hcy and cysteine (each
at 0.3 mM) [434].
The cause of connective tissue abnormalities resulting from hyperhomocys-
teinemia has been studied in an in vivo chick model [19]. Hyperhomocysteinemia
was induced by feeding 2-day-old chicks with a high-Met diet for up to 9 weeks.
The aortas of the hyperhomocysteinemic chicks show severe histopathology,
including pronounced separation of elastic lamellae with marked smooth muscle
proliferation and aneurysms. Electron microscopy studies revealed disordered
elastic fibers in aorta and absence or disrupted assembly of microfibrils. Immuno-
histochemical examinations demonstrated a loss of fibrillin-2 in the aortic tissue.
Although it has not been examined whether these abnormalities are linked to
S-homocysteinylation of any of the connective tissue components, these data
provide evidence that elevated Hcy or its metabolites disrupt normal microfibril
structure, leading to aberrant elastic fibers [19].