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Schools of Medicine, Dentistry and Health Related Professions, Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-3360
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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KEY WORDS: • homocysteine • homocysteine thiolactone • protein homocysteinylation • aging • fibrillin-1 • EGF-like domains
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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).
Higher levels define hyperhomocysteinemia which, if severe (>10 times
normal) as in the rare inborn error of metabolism homocystinuria, is
accompanied by a plethora of serious clinical manifestations occurring
early in life. Primarily affected are the eye and three organ systems,
i.e., the skeletal, vascular and central nervous systems. Severe myopia
and dislocation of the lens, osteoporosis together with skeletal
malformations, often lethal thromboembolic vascular occlusions of
arteries and veins, and mental impairment develop in childhood or young
adulthood (Mudd et al. 1995
). A large body of
epidemiologic evidence now supports the notion that similar clinical
counterparts develop many years later in normal individuals as part of
the aging process.
Moderate hyperhomocysteinemia (tHcy up to 30 µmol/L)
(Kang et al. 1992
), a common condition, is a major
independent risk factor for a number of diseases characteristic of old
age, primarily occlusive vascular disease (coronary, cerebral and
peripheral) (Boushey et al. 1995
) cognitive decline,
including Alzheimer’s disease (Clarke et al. 1998
), and
possibly, senile osteoporosis (Miyao et al. 1998
) and
presbyopia (Krumdieck, unpublished). These disorders, which together
account for much of the morbidity and mortality in the aged, are
strikingly similar in all aspects but time of onset, to the
main manifestations of homocystinuria and could be considered as the
clinical signs of a single disorder of late life, i.e., chronic
moderate hyperhomocysteinemia. The contribution of chronic moderate
hyperhomocysteinemia to diseases of old age may have gone unrecognized
because we are conditioned to accept them as inescapable consequences
of growing old. The possibility of lessening the effect of
hyperhomocysteinemia as a determinant of premature aging by appropriate
nutritional interventions known to lower tHcy, i.e., supplemental
folate, vitamins B-6 and B-12, and reducing the intake of methionine,
could change this perception.
The role of Hcy in aging is supported by the experimental studies of
Orentreich et al. (1993)
and Richie et al. (1994)
on the extension of life span in rats brought about by
diets severely restricted in methionine and devoid of cysteine. A 42%
increase in mean life span and a 44% increase in maximum life span was
observed in these rats compared with controls. Under these dietary
conditions, the only route to cysteine synthesis in mammals, the
transulfuration of Hcy and serine through the cystathionine ß
synthase reaction, and the reactions of regeneration of methionine by
remethylation of Hcy catalyzed by methionine synthase and by
betaine:homocysteine methyltransferase, would be completely
de-repressed (Mudd et al. 1995
). All Hcy formed
in these rats must have been converted immediately to cysteine or
remethylated to methionine, resulting in extremely low levels of tHcy
in circulation. Unfortunately, tHcy was not reported and the authors
did not discuss the possible contribution of the lowering of tHcy to
the observed effects on life span.
As aptly put by Mudd et al. (1995)
, "... no
aspect ... has remained so obscure as the steps by which
hyperhomocysteinemia leads to the clinical manifestations associated
with it." No single mechanism that by itself explains
the multisystem toxicity of hyperhomocysteinemia has been proposed,
maybe because no single mechanism can. Ironically, it
may be that its baffling multiplicity of actions contains a valuable
hint to the mechanism of Hcy toxicity. That is, Hcy reacting with
proteins by one or a few reactions, may damage the components of many
metabolic pathways, thus acquiring pleiotropic toxicity.
Homocysteine, a short-lived highly reactive thiol-containing
amino acid formed as an obligatory intermediate in the metabolism of
methionine, belongs to the most chemically active group of compounds
found in the animal organism (Jocelyn 1972
). It is
capable of reacting specifically, and often quantitatively, with a
number of thiol-combining groups, many of which are present in
proteins and other biologically important molecules. In addition, Hcy
cyclizes readily with the formation of Hcy thiolactone (Hcytl), an
"activated" intramolecular thioester with its own unique repertoire
of chemical reactions (Dudman et al. 1991
). It has been
proposed that the harmful effects of Hcy and Hcytl are due to their
spontaneous (i.e., nonenzymatic) chemical reactions with, and
inactivation of proteins and other biologically important
molecules. Free Hcy readily adds to free,
solvent-accessible, cysteinyl residues in proteins (thiol-thiol
interaction) and can cleave solvent-accessible disulfide bridges
(thiol-disulfide exchange) with damage to the folding pattern of the
protein. Homocysteine thiolactone, whose long-questioned formation
in vivo has now been elegantly demonstrated by Jakubowski (1997
and 2000
), reacts through its carboxyl group with the
-NH2 group of lysyl residues with the irreversible
formation of homocystamide derivatives, as first shown more than 40
years ago by Benesch and Benesch (1956)
. All of these
reactions of protein homocysteinylation can lead to loss or degradation
of the biological function of multiple enzymes, receptors, growth
factors and structural proteins and can be envisioned as analogous to
protein glycation, the reaction of proteins with glucose in protracted
hyperglycemia, believed to cause many of the complications of diabetes
(Brownlee 1992
). It should be noted that protein
homocysteinylation occurs normally in vivo at physiologic
concentrations of tHcy, as first shown for albumin by Kang et al. (1979)
and recently by Hajjar et al. (1998)
and Ling and Hajjar (2000)
who also demonstrated a
concomitant loss of biological activity of the derivatized protein,
annexin II.
As is true for any other chemical reaction, the extent of formation of Hcy and Hcytl protein derivatives is dependent on time and concentration. The longer the duration of exposure and the higher the concentrations of Hcy or Hcytl, the greater the biochemical damage inflicted. Furthermore, if the molecules attacked are long-lived and the derivation reactions irreversible (i.e., formation of homocystamide), the harmful effects will be cumulative and the clinical consequences progressive.
It is of fundamental importance to investigate which proteins are
particularly susceptible to Hcy and Hcytl attack. A good criterion to
select likely targets for homocysteinylation is to focus on proteins
found in structures singularly affected in homocystinuria. One such
structure is the ciliary zonule (the suspensory ligament of the lens),
a small anatomical structure which, if damaged, results in dislocation
of the lens. This rare clinical manifestation occurs mainly in
homocystinuria and in Marfan’s syndrome (Pyeritz 1993
),
a disorder of connective tissue which, aside from lens dislocation, has
skeletal and cardiovascular abnormalities similar to those of
hyperhomocysteinemia. The zonule is formed solely by
elastic microfibrils (Cleary and Gibson 1996
), which
forces the conclusion, verified by histopathologic demonstration of
fraying and disruption of the zonula fibers (Mudd et al. 1995
), that it is these fibers and hence their constitutive
proteins that must be damaged in both homocystinuria and Marfan’s
syndrome. Focusing on one zonular protein was made possible by the
recent identification of fibrillin-1 as the main component of elastic
microfibrils (Sakai et al. 1991
). Fibrillin-1, a large
rod-like glycoprotein with an exceptionally high cysteine content
(~14%) much of which (~33%) appears in the free reactive
sulfhydryl form, is also found in the medial layer of all elastic
arteries, in the heart, bone, periostium, cartilage, skin and lung, all
structures that are compromised in both Marfan’s syndrome and
homocystinuria. Recently, mutations in the fibrillin-1 gene have been
shown to cause Marfan’s syndrome (Dietz et al. 1991
,
Lee et al. 1991
), supporting the assumption that
fibrillin-1 is an important target of homocysteinylation and that the
resulting post-translational damage to its structure is responsible
for many of the abnormalities that severe hyperhomocysteinemia has in
common with Marfan’s syndrome.
Fibrillin-1 consists largely of 56 cysteine-rich imperfect repeat
domains, 47 of which show significant homology to a motif originally
found in epidermal growth factor (EGF), the "EGF-like" repeats
(Cleary and Gibson 1996
). These repeats are
characterized by six predictably spaced cysteine residues that interact
to form three highly critical disulfide bonds. No conserved hydrophobic
residues are required to maintain the EGF-like domain fold; it is
stabilized by the three disulfide bonds, which form in a 1–3, 2–4,
5–6 pattern (Bork et al. 1996
). The biological
significance of these cysteinyl residues is demonstrated by the fact
that many of the mutations causing Marfan’s syndrome substitute one of
the highly conserved cysteinyl residues, add a new one, or alter their
relative spacing. Many of the EGF-like domains of fibrillin-1
contain a consensus for Ca++ binding (cb), a property that
may contribute to stabilize the disulfide bonds of these cb-EGF-like
modules. Free homocysteine may react with some of the numerous free
cysteinyl sulfhydryl (SH) groups in fibrillin-1 or may disrupt
critical Cys-Cys disulfide bridges in EGF-like domains with the
formation of protein mixed disulfides via thiol-disulfide exchange
reactions.
Fibrillin-1 may also be irreversibly homocysteinylated at lysyl residues, many of which are concentrated in a long (17,587-Da) region at the carboxyl end of the protein, which is conspicuously lacking in cysteinyl residues. This region may be involved in intermolecular fibrillin aggregation. Its homocysteinylation may therefore impede the formation of microfibrils.
Alternatively, the insertion of an amide-linked homocysteine
introduces a new free SH group that can react with an adjacent
disulfide bridge in an intramolecular thiol-disulfide exchange
reaction. A native disulfide bridge could be cleaved and a new mixed
disulfide bridge formed between one of the cysteinyl residues and the
aberrant Hcy-lysyl-amide side chain. A similar reaction involving a
free cysteine SH group has been described in albumin (Jocelyn 1972
and references therein). Disulfide cleavage, with the
major disruption in folding it brings about, may thus result from the
introduction of an amide-linked homocysteinyl residue.
It is worth noting that fibrillin-1 is a long-lived protein, which in some locations, such as the ciliary zonule, may be as old as the individual. In addition, the zonule, bathed by the essentially albumin-free aqueous humor, may be devoid of the protection against homocysteinylation reactions conferred in other sites by the presence of albumin, which, with its highly reactive free cysteine 34 and its high proportion of lysyl residues, may act as an efficient scavenger of both free Hcy and Hcytl.
Further support for the proposed mechanism of protein damage involving
cleavage of disulfide bridges is provided by the similarity of the
clinical manifestations of severe hyperhomocysteinemia with those of
Sulfite Oxidase and Molybdenum Cofactor deficiencies. In these two rare
disorders, the sulfite anion (-SO3H)
accumulates and reacts with cysteine to form sulfocysteine. As in
severe hyperhomocysteinemia, ectopia lentis, skeletal malformations and
vascular occlusions develop early in life (Johnson and Wadman 1989
). Accumulation of either homocysteine or
-SO3H, both of which react avidly with thiols
and cleave disulfides bridges by thiol-disulfide exchange and
sulfitolysis, respectively, produces similar symptoms and discloses
commonality of pathogenetic mechanisms.
The hypothesis that the EGF-like domains of fibrillin-1 are
specially vulnerable sites of homocysteinylation suggests that other
extracellular proteins with similar regions could also be particularly
susceptible to Hcy attack. The presence of EGF-like domains in many
proteins involved in the pathways of coagulation (factors V, VII, IX,
X, thrombin receptor, thrombin activatable fibrinolysis inhibitor),
anticoagulation (protein C, thrombomodulin, protein S, antithrombin
III), thrombolysis (plasminogen activator) and lipoprotein
translocation (several lipoprotein receptor molecules) is highly
significant because thrombophilia and premature arteriosclerosis are
prominent manifestations of hyperhomocysteinemia. Of particular
importance the protein C/thrombomodulin/protein S system is made up of
proteins that contain EGF-like modules. Their susceptibility to
inactivation by Hcy was demonstrated in vitro as far back as 1991
(Lentz and Sadler 1991
). These authors showed that free
Hcy, but not homocystine, irreversibly destroys the biological activity
of both thrombomodulin and protein C, preventing the activation of the
latter by thrombomodulin-modified thrombin. The normal degradation
of activated coagulation factors Va and VIIIa by activated protein C,
which prevents the catastrophic progression of the coagulation cascade,
would fail to occur, leading to thrombus formation (Dahlback 1995
). Several other anti- and procoagulant proteins such as
antithrombin III, plasminogen activator, thrombin receptor and Hageman
factor (the latter activated by Hcy) also contain EGF-like domains
and hence are suspected homocysteinylation targets.
It is of great interest that the amino-terminal region of the LDL
receptor (LDLR) consists of seven tandem repeated cysteine-rich
modules of ~40 amino acids (the LDL-A modules) each of which
contains six cysteine residues disulfide-bonded in a manner similar
to the EGF-like domains (Brown et al. 1997
,
Fass et al. 1997
). Reduction of these disulfides
destroys the structure and abolishes binding of the LDL particle to the
receptor (Brown et al. 1997
). Genetic mutations leading
to this condition cause familial hypercholesterolemia with its
accompanying arteriosclerosis. The structure of the LDL-A modules
is stabilized not only by the disulfide bridges but also by a single
atom of Ca++ contained in an octahedral cage. In this
manner the LDL-A modules resemble also the calcium-binding
EGF-like domains of the fibrillins. The important notion is that
homocysteinylation of LDLR may lead to inactivation of the receptor and
provide an explanation for the propensity to develop arteriosclerosis
in hyperhomocysteinemia.
It is important to consider also the effect that different amino acid
sequences will have on the susceptibility to Hcy or Hcytl attack of
different proteins. It can be expected that some EGF-like domains
will be more susceptible to attack than others. Genetic polymorphisms
of EGF-like domains or other regions of susceptible proteins will
determine their lability to attack and help explain the association
between risk of pathology and a wide range of tHcy levels without a
clear cut-off point below which there is no increased risk
(Verhoef et al. 1997
).
In addition to protein homocysteinylation, Hcy modifies the release or
activity of small molecules of endothelial origin, primarily nitric
oxide, that have pronounced vasoactive effects in response to blood
flow (Bellamy and McDowell 1997
, McDowell and Lang 2000
) or that can modulate cell proliferation and
differentiation in blood vessels (Dalton et al. 1997
).
Oxidative damage produced by homocysteine-mediated generation of
free radicals is yet another possible mechanism of Hcy toxicity. None
are mutually exclusive and, indeed, all may be involved simultaneously.
Finally, a word of caution regarding the methodologies for
determination of homocysteine status. Current procedures for measuring
tHcy rely on the assumption that all forms of
protein-bound Hcy in circulation are liberated after reduction.
This is incorrect; amide-linked Hcy is completely stable to
reductive cleavage, and no method for the routine quantitation of this
adduct in plasma or in tissues is currently available. Given the
potential pathologic significance of protein homocysteinylation at
lysyl residues discussed before and exemplified by the irreversible
inhibition of lysyl oxidase by Hcytl (Liu et al. 1997
),
every effort should be directed to the development of the required
methodologies.
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FOOTNOTES |
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3 Abbreviations used: cb, Ca++ binding; EGF, epidermal growth factor; Hcy, homocysteine; Hcytl, Hcy thiolactone; LDLR, LDL receptor; SH, sulfhydryl; tHcy, total Hcy in plasma.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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