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Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA
1Correspondence: Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. E-mail: jakubows{at}umdnj.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: homocysteine thiolactone • atherosclerosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, it is not known why Hcy is
harmful. Among the possible aspects of Hcy metabolism that may account
for detrimental effects of elevated Hcy levels, conversion to Hcy
thiolactone as a result of an error-editing function of some
aminoacyl-tRNA synthetases (AARS in Eq. 1
) has been
extensively studied (2
3
4
5
6
7)
.
 |
) (8
, 9)
.
 |
,
detailed studies of reactions of Hcy thiolactone with proteins were
undertaken and are reported here. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and purified by 2D thin-layer
chromatography (TLC) on cellulose plates (20 x 10 cm; Kodak)
(7)
. The overall yield of the procedure was 65%. The
preparation of L-[35S]Hcy
thiolactone was at least 96% pure on analytical 2D TLC. Maximum levels
of contamination with Met, Hcy, and homocystine were <1%, <0.8%,
and <1%, respectively.
Proteins
Methionyl-tRNA synthetase (MetRS) was purified to homogeneity
from an overproducing strain of Escherichia coli as
described before (5)
. Other pure proteins and sera were
purchased from Sigma.
Reactions of homocysteine thiolactone with proteins
Unless otherwise stated, reactions were carried out at 37°C in
mixtures containing L-[35S]Hcy
thiolactone, 10 mg/ml protein, 0.1 M sodium phosphate buffer (pH 7.4),
0.2 mM EDTA. Phosphate buffer and EDTA were excluded when reactions of
proteins in serum with Hcy thiolactone were studied;
L-[35S]Hcy thiolactone (1% volume)
was added directly to serum. Formation of homocysteinylated proteins
was determined by precipitation with 5% trichloroacetic acid (TCA)
and/or by TLC (7)
. Both methods gave identical results.
Homocysteinylated proteins were also analyzed by electrophoresis on
native and denaturing polyacrylamide gels under non-reducing and
reducing conditions according to standard protocols (11)
.
Edman degradation
Automated Edman degradation was carried out by Dr. R. Donnelly
on a model 491 ProciseTM Applied Biosystems
protein sequencer in the New Jersey Medical School Molecular Resource
Facility.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), ~25-fold faster than the rate of non-enzymatic
hydrolysis of the thiolactone (7)
. At the end of
incubation at 3 h, most of the thiolactone was incorporated
covalently into protein. Addition of dithiothreitol (DTT) to
[35S]Hcy thiolactone-modified serum resulted in
a release of ~50% of the incorporated 35S as
free [35S]Hcy. The DTT-resistant fraction of
35S-protein adduct was also resistant to
treatments with 0.1 M NaOH, 1 M hydroxylamine, or rabbit esterase (all
of which destroy ester bonds). To confirm that DTT treatment releases
all Hcy from protein-S-S-Hcy disulfide adducts,
[35S]Hcy (2–300 µM) was incubated with human
serum to form protein-S-[35S]Hcy adducts. All
[35S]Hcy became protein-bound after 2 h at
37°C as determined by TCA precipitation and TLC analyses. DTT
treatment of these adducts rendered all radioactivity TCA-soluble; only
free [35S]Hcy was observed after TLC analysis
of the DTT-treated adducts (not shown). These results indicate that Hcy
thiolactone undergoes two major reactions in serum: 1) one
reaction involves acylation of amino groups in proteins; 2)
the other reaction involves enzymatic hydrolysis (7
, 12)
to give Hcy, which then attaches to proteins by forming a
protein-S-S-Hcy disulfide.
|
As shown in Figure 1B
, homocysteinylation of human serum
proteins was observed at thiolactone concentrations as low as 10 nM and
was directly proportional to concentrations of Hcy thiolactone up to 1
mM.
DTT-resistant 35S-protein adducts were obtained
with serum samples from 10 different human donors and with a range of
[35S]Hcy thiolactone concentrations from 10 nM
to 5 mM (Table 1
). Kinetics of protein homocysteinylation and thiolactone hydrolysis in
bovine and horse sera (not shown) were similar to those observed in
human serum shown in Figure 1A
. However, in rabbit and mouse
sera Hcy thiolactone disappeared with a half-life of 15 min (Table 1)
,
some fourfold faster than in human serum. Only ~10% of the initially
present [35S]Hcy thiolactone was incorporated
into rabbit or mouse serum proteins (Table 1)
.
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Individual proteins present in serum can be homocysteinylated
To determine which proteins in serum can be homocysteinylated,
sera were incubated for 2 h with
[35S]thiolactone and subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing conditions (1% 2-mercaptoethanol). As shown in Figure 2
, many proteins present in human and rabbit sera were radiolabeled. It
appears that each protein was radiolabeled roughly in proportion to its
abundance in serum: Coomassie blue-stained serum protein gels (not
shown) and their corresponding autoradiographs (Fig. 2)
showed
identical patterns and similar relative intensities of protein bands.
Bands of major serum proteins, albumin, 
-globulin, fibrinogen,
transferrin, and 
2-macroglobulin, were the
most heavily 35S-labeled. These proteins were
identified by their co-migration with authentic standards of individual
pure proteins.
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Kinetics of protein homocysteinylation
Measurements of homocysteinylation rates showed that total protein
in human serum was modified only 30% faster than an equivalent
concentration of pure human albumin, which in turn was
homocysteinylated about as fast as an equivalent concentration of free
lysine. This suggests that protein homocysteinylation in human serum is
due to a non-enzymatic reaction.
Second-order rate constants for homocysteinylation of individual
proteins (Table 2
) indicate that reactions of Hcy thiolactone with proteins are robust
and go to completion within a few hours under physiological conditions
of pH and temperature. It appears that a major determinant of the
reactivity of most proteins is their lysine content. For example, Hcy
thiolactone reacts with poly-lysine two to three orders of magnitude
faster than with most proteins. For proteins that vary in size from 104
to 698 amino acid (AA) residues, there is a very good correlation (with
a correlation coefficient of 0.97) between protein lysine contents and
their reactivities with the thiolactone (Fig. 3
). A weaker correlation (with a correlation coefficient of 0.75) is
observed between the size of proteins and their reactivities toward the
thiolactone (not shown). However, larger proteins such as fibrinogen
(3588 AA residues), low-density lipoprotein (LDL; ~5000 AA residues),
and 2-macroglobulin (5896 AA residues) are
homocysteinylated much less efficiently than expected from either
their lysine contents or sizes. Apparently, a major fraction of lysine
residues of these proteins is not accessible to solvent.
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Homocysteinylation leads to loss of function and denaturation
Effects of homocysteinylation on protein function were examined.
Two model enzymes, MetRS and trypsin, were homocysteinylated to
different degrees with the thiolactone and their enzymatic activities
were determined. There was a progressive loss of enzymatic activity
with increasing degree of homocysteinylation (Fig. 4
). MetRS was fully inactivated when eight to nine Hcy residues were
incorporated per molecule of the enzyme (corresponding to ~33%
lysine residues modified). Trypsin was fully inactivated when 11–12
Hcy residues were incorporated per molecule of the protein (~88%
lysine residues modified).
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Structural effects of homocysteinylation were assessed by analyzing
behavior of modified proteins on native and denaturing polyacrylamide
gels. Extensively homocysteinylated albumin, -globulin, fibrinogen,
transferrin, and 2-macroglobulin migrated
faster than the corresponding less extensively modified proteins on
native PAGE gels (Fig. 5
). This is consistent with an increase in negative charge of the
modified proteins after homocysteinylation of lysine residues. Because
the 
-amino group of lysine (pK=10.5) is much more basic
than the -amino group of Hcy in N(Hcy)lysine
(pK=7.1) the positive charge of lysine residues is lost
after homocysteinylation. In addition to increased electrophoretic
mobility, denaturation of the homocysteinylated proteins was also
observed: a fraction of each homocysteinylated protein remained in the
wells and did not enter the gel (lanes 7–12 in Fig. 5
). The denatured
homocysteinylated proteins were also observed in the wells of SDS-PAGE
gels (not shown).
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Homocysteinylation results in incorporation of additional -SH groups
into proteins. Spontaneous oxidation of these thiols could lead to
formation of intermolecular disulfide bonds and formation of protein
multimers. Indeed, homocysteinylation of cytochrome c with
increasing concentrations of thiolactone resulted in formation of
cytochrome c dimers, trimers, tetramers, etc. (Fig. 6
). These multimers disappeared when modified cytochrome c was
treated with 2-mercaptoethanol before loading onto SDS-PAGE gel (not
shown).
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To determine a relationship between the degree of homocysteinylation
and protein denaturation, solubility of the modified proteins was
studied. For each of the tested proteins there was a threshold of
homocysteinylation after which proteins began to precipitate
(Fig. 7
). Some proteins were more affected by homocysteinylation than others.
For example, trypsin (Fig. 7A
) and RNase A (Fig. 7B
) began to precipitate when only ~1 mole Hcy was
incorporated per mole protein. Hemoglobin (Fig. 7B
) also
began to precipitate at a similar low level of homocysteinylation;
however, whereas further increase in homocysteinylation led to a
precipitous loss of solubility of trypsin and RNase A, there was a less
extensive gradual loss of solubility of hemoglobin. When the degree of
homocysteinylation exceeded 5 mol Hcy/mol protein, myoglobin and
cytochrome c (Fig. 7B
) began to precipitate. With
the maximum number of 14 mol Hcy/mol protein (74% lysines modified),
cytochrome c was still 50% soluble. Some proteins, such as
fibrinogen, albumin, -globulin (Fig. 7A
), and transferrin
(Fig. 7B
), remain fully soluble with as many as 20 mol Hcy
incorporated/mol protein but eventually began to precipitate at higher
levels of homocysteinylation.
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Homocysteine can be recovered from homocysteinylated proteins
To exclude the possibility that Hcy thiolactone undergoes some
unexpected reaction with proteins, as opposed to expected direct
acylation of protein lysine residues, attempts were made to determine
whether Hcy can be recovered from homocysteinylated proteins. For these
experiments, samples of serum as well as purified human serum albumin,
homocysteinylated with either unlabeled or
35S-labeled Hcy thiolactone, were used.
In one series of experiments, [35S]homocysteinylated serum proteins (containing 40 µM covalently incorporated Hcy) from which low-molecular-weight thiols were removed by repeated treatments with DTT and precipitations with ethanol, were subjected to acid hydrolysis with HCl/propionic acid in the presence of 3-mercaptopropionate. After hydrolysis (4 h, 80°C), Hcy was recovered as thiolactone, with 80% yield. When acid hydrolysis was carried out in the absence of 3-mercaptopropionate, homocystine was recovered.
A similar sample of homocysteinylated serum proteins, containing 50 µM covalently incorporated non-radiolabeled Hcy and depleted of low-molecular-weight thiols, was treated with 4-vinylpyridine to protect the -SH group of Hcy. Subsequent Edman degradation on an amino acid sequencer yielded S-(4-ethylpyridine) derivative of phenylthiohydantoin-Hcy (PTH-Hcy) in the first cycle, as expected. A sample of homocysteinylated human serum albumin, containing 3 mol Hcy/mol protein, was alkylated with iodoacetate to protect the -SH group of Hcy. The resulting S-(carboxymethyl) derivative of PTH-Hcy was recovered in the first cycle of Edman degradation, eluting at a position of glutamic acid, as expected. Another major product was PTH-Asp, corresponding to the amino-terminal aspartate of human albumin. Quantitation of chromatographic peaks corresponding to these two products showed that the amount of Hcy was three times greater than the amount of Asp. This result suggests that the amino-terminal Asp is not a major site of homocysteinylation in human albumin; if it were, the amino-terminal Asp would not be recovered.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because Hcy forms as a byproduct of methylation reactions in human
cells, metabolic conversion of Hcy to the thiolactone is inadvertent
and occurs to a lesser or greater extent depending on specific genetic
and metabolic conditions. The extent of thiolactone synthesis depends
on relative levels of Hcy and Met, the levels of expression and
activity of MetRS, methionine synthase, and cystathionine ß-synthase
(2
3
4
5
6
7)
. Because the activity of major Hcy-metabolizing
enzymes requires cofactors such as vitamin B12
and folate (methionine synthase) as well as vitamin
B6 (cystathionine ß-synthase), the extent of
thiolactone synthesis also depends on the levels of these vitamins.
Mutations in genes encoding methionine synthase (3)
and
cystathionine ß-synthase (3
, 7)
lead to enhanced
metabolic conversion of Hcy to the thiolactone. In the extreme cases
when the function of Hcy-metabolizing enzymes is abolished, all Hcy is
converted to the thiolactone (3
, 6
; H. Jakubowski,
unpublished data). These effects on Hcy thiolactone synthesis occur in
all cell types investigated, from bacterial to human
(2
3
4
5
6
7)
.
Regarding human cells, cystathionine ß-synthase-deficient
fibroblasts produce more Hcy thiolactone than unaffected cells.
Inhibition of methionine synthase by an antifolate drug, aminopterin,
leads to enhanced synthesis of Hcy thiolactone in normal human
fibroblasts as well. Human umbilical vein endothelial cells limited for
vitamin B12 and folate synthesize micromolar
concentrations of Hcy thiolactone (H. Jakubowski, unpublished data).
Concentrations of the thiolactone in tissue cultures are about
one-tenth the concentration of Hcy (7)
.
Hcy thiolactone easily reacts with proteins under physiological
conditions. In tissue cultures of human fibroblasts cellular proteins
become homocysteinylated (7)
. Recent experiments with
cultured human endothelial cells show that both cellular and
extracellular proteins become extensively homocysteinylated under
conditions of vitamin B12 and folate limitation.
The concentration of homocysteinylated proteins can reach up to 40 µM
within 48 h under these conditions (H. Jakubowski, unpublished
data). In in vitro experiments with human serum spiked with
[35S]Hcy thiolactone, the thiolactone
disappeared with a half-life of ~1 h, and each serum protein became
homocysteinylated. Protein homocysteinylation in human serum occurs at
as low as 10 nM thiolactone and increases directly in proportion to the
increase in the thiolactone concentration, up to millimolar range.
Thus, regardless of how small or large quantities of Hcy thiolactone
are made, the thiolactone modifies proteins. If conditions favoring
synthesis of Hcy thiolactone, such as elevated levels of Hcy, are
maintained, there will be a concomitant increase in the degree of
protein homocysteinylation.
Chemical reactivities of homocysteinylated proteins as well as kinetics
of protein homocysteinylation reported here suggest that side chain
amino groups of lysine residues are the major sites of
homocysteinylation in most proteins. For example, Hcy thiolactone
reacts with most proteins at rates similar to the rate of the reaction
with an equivalent concentration of lysine or
-N-acetyl-lysine (Table 2)
. This explains why protein
homocysteinylation correlates better with protein lysine content than
with protein size (Fig. 3)
. Homocysteinylated proteins do not release
Hcy on treatments with agents that hydrolyze ester bonds. However, Hcy
is released from homocysteinylated proteins by acid hydrolysis or Edman
degradation. The possibility that side chains of histidine and arginine
could also be modified is unlikely because free arginine or
-N-acetyl-histidine do not appreciably react with Hcy
thiolactone (not shown).
Homocysteinylation can lead to protein damage. As demonstrated here,
homocysteinylation of 33% of lysine residues in MetRS and 88% lysine
residues in trypsin resulted in complete loss of their enzymatic
activities. Homocysteinylated proteins were prone to multimerization
and underwent gross structural changes that led to their denaturation.
Homocysteine thiolactone may also inactivate enzymes by other
mechanisms. For example, lysine oxidase, an important enzyme
responsible for posttranslational collagen modification essential for
the biogenesis of connective tissue matrices, is inactivated by Hcy
thiolactone, which derivatizes the active site tyrosinequinone cofactor
(14)
.
In addition to a loss of function, protein homocysteinylation can also
generate modified proteins that are physiologically detrimental in
other ways. For example, homocysteinylated LDL has been recently shown
to elicit immune response in rabbits (15)
. Rabbit
antiserum against an Hcy-LDL adduct was also shown to react with
adducts of Hcy and other proteins, such as bovine serum albumin,
hemoglobin, and serum proteins. This antigen specificity suggests that
the rabbit antiserum reacts with Hcy-Lys-epitopes (15)
.
How can protein damage lead to cell injury, a hallmark of atherosclerosis? One plausible scenario is that homocysteinylated proteins on the surface of vascular vessels will be recognized by macrophages either directly or indirectly. Macrophages will attempt to phagocytize damaged proteins on the surface of endothelial cells, which would lead to destruction of endothelial cells and damage to vascular wall. Alternatively, homocysteinylated endothelial cells will attract anti-Hcy-protein antibodies and form antigen-antibody complexes on the surface of vascular vessel. Endothelial cells coated with antibody will be recognized and then bound by macrophages through their Fc receptors. After binding, the endothelial cells will be ingested and destroyed, which will result in injury to the vascular surface. If the agent responsible for the injury (homocysteinylated proteins) is present continuously, attempts to repair the damaged vascular wall will eventually lead to an atherosclerotic plaque.
Protein homocysteinylation is a novel example of protein damage that
may explain the involvement of Hcy in the pathology of human vascular
diseases. However, other protein modifications by drugs or cellular
metabolites have been implicated in other human diseases. For example,
acetylation of proteins by aspirin is thought to be an underlying cause
of aspirin intolerance (16)
. Peniciloylation of proteins
by the antibiotic penicillin is involved in penicillin allergy
(17)
. Modification of proteins by glucose is believed to
underlie the pathogenesis of diabetes (18)
and
Alzheimer’s disease (19)
. Proteins modified by products
of lipid oxidation are implicated in the etiology of atherosclerosis
(20)
. A common aspect of these modification reactions is
the involvement of protein lysine residues as sites of
modifications.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and the disappearance of Hcy thiolactone (
).
B) Relationship between concentration of Hcy thiolactone
and the degree of protein homocysteinylation for sera from two
different subjects (•, 
) and trypsin (•) are plotted as a function of a number of Hcy
molecules incorporated per molecule of protein.
);
(B), transferrin (
), and RNase A (