Homocysteine Thiolactone and Protein Homocysteinylation

in Human Endothelial Cells

Implications for Atherosclerosis
Hieronim Jakubowski, Li Zhang, Arlene Bardeguez, Abram Aviv
AbstractEditing of the nonprotein amino acid homocysteine by certain aminoacyl-tRNA synthetases results in the
formation of the thioester homocysteine thiolactone. Here we show that in the presence of physiological concentrations
of homocysteine, methionine, and folic acid, human umbilical vein endothelial cells efficiently convert homocysteine
to thiolactone. The extent of this conversion is directly proportional to homocysteine concentration and inversely
proportional to methionine concentration, suggesting involvement of methionyl-tRNA synthetase. Folic acid inhibits the
synthesis of thiolactone by lowering homocysteine and increasing methionine concentrations in endothelial cells. We
also show that the extent of post-translational protein homocysteinylation increases with increasing homocysteine levels
but decreases with increasing folic acid and HDL levels in endothelial cell cultures. These data support a hypothesis that
metabolic conversion of homocysteine to thiolactone and protein homocysteinylation by thiolactone may play a role in
homocysteine-induced vascular damage. (Circ Res. 2000;87:45-51.)
Key Words: homocysteine proteins HDL lipoproteins endothelial cells atherosclerosis

levated levels of the nonprotein amino acid homocysteine (Hcy)1 are associated with vascular disease in
humans.1 However, it is not known why Hcy can be harmful.
The conversion of Hcy to thiolactone as a result of an
error-editing function of some aminoacyl-tRNA synthetases
(AARS in Equation 1)2,3 is one feature of Hcy metabolism
that may account for detrimental effects of elevated Hcy
levels.
(1)

Hcy thiolactone, originally discovered in cultures of microbial cells,2 4,1113 is also synthesized by cultured mamma-

(2)

AARSHcyATP N AARSHcyAMPPPi
2

lian nonvascular cells.8,14 Whereas methionyl-tRNA synthetase is involved in synthesis of thiolactone in all cell types
examined,1114 isoleucyl- and leucyl-tRNA synthetases can
also convert Hcy to thiolactone, at least in bacteria.13 Because
of its mostly neutral character at physiological pH (pK7.1;
Reference 15), thiolactone accumulates in culture media.8,1114 Small amounts of Hcy are present in proteins.2,8,9
It is not known whether Hcy thiolactone synthesis occurs
in human vascular endothelial cells. If it did, this could
provide a plausible chemical mechanism explaining Hcy
toxicity to the human vascular endothelium, the damage of
which plays a central role in atherosclerosis.16 Human umbilical vein endothelial cells (HUVECs), used frequently as a
model of vascular cells, have been reported to possess Hcy
thiolactone hydrolyzing activity17 and therefore are thought

Hcy thiolactone
This conversion involves reaction of Hcy with ATP to form
an AARS-bound homocysteinyl adenylate (HcyAMP) and
inorganic pyrophosphate, PPi. Misactivated Hcy is not transferred to tRNA by any of these synthetases.37 Instead,
HcyAMP undergoes a reaction in which the side-chain thiol
group (SH in Equation 2) of Hcy displaces the AMP group
from the carboxylate of the activated Hcy, forming Hcy
thiolactone as a product (Equation 2).5,7 The energy of the
anhydride bond of HcyAMP is conserved in an intramolecular thioester bond of thiolactone. Consequently, Hcy
thiolactone is chemically reactive and acylates free amino
groups, such as side-chain lysine groups in proteins.8 10
Homocysteinylated proteins lose their biological activity.10

Received April 27, 2000; accepted May 10, 2000.

From the Department of Microbiology and Molecular Genetics (H.J.), Hypertension Research Center (L.Z., A.A.), and Department of Obstetrics and
Gynecology (A.B.), UMDNJ-New Jersey Medical School, Newark, NJ.
Presented in preliminary form at the Experimental Biology 99 meeting, Washington, DC, April 1721, 1999, at the symposium Homocysteine, Aging
and Geriatric Disease.
Correspondence to Hieronim Jakubowski, Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 185 S Orange
Ave, Newark, NJ 07103. E-mail jakubows@umdnj.edu
2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org

45

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Circulation Research
TABLE 1.

July 7, 2000

Intracellular and Extracellular Levels of [35S] Metabolites in HUVECs

Culture Conditions

Thiolactone

5 mol/L Met

Homocysteine

Methionine
3.40.6 (80) [3%]

0.03 (50) [2%]

0.3 (350) [13%]

0.01 (5.0) [0.2%]

0.06 (30) [1.2%]

2.90.1 (90) [3.6%]

20 mol/L Met

0.01

1.0

24 (5000)

2 mol/L Hcy

0.050.01 (50) [5%]

0.750.12 (900) [90%]

0.230.06 [0.02%]

8 mol/L Hcy

0.270.01 (250) [6%]

4.71.2 (3250) [80%]

0.670.05 [0.015%]

0.01 (20) [0.5%]

2.6 (3000) [75%]

2.6 [0.06%]

5 mol/L Met10 mol/L folic acid

8 mol/L Hcy10 mol/L folic acid

5

Data are presented as pmol/10 cells. Values in parentheses represent extracellular concentrations. Bracketed values are
percentages of total [35S] present in indicated metabolites.

not to be able to support thiolactone synthesis. However, in

this work, we found that cultured HUVECs efficiently metabolize Hcy to thiolactone and provide evidence of posttranslational incorporation of Hcy into proteins in these
cultures. We also examined factors affecting thiolactone
synthesis and protein homocysteinylation in HUVECs.

Materials and Methods

Cell Culture
HUVECs were obtained from umbilical cords of 6 healthy newborns.
Each cord was separately harvested in a solution containing
(in mmol/L) HEPES (pH 7.3) 10, NaCl 140, KCl 4, and glucose 11;
50 g/mL penicillin; and 100 g/mL streptomycin (Sigma). Endothelial cells were isolated by collagenase treatment18 within 4 to 5
hours after delivery. Cells were seeded into 75-cm2 culture flasks
coated with collagen and cultured in M199 (Life Technologies, Inc)
at 37C/5% CO2, supplemented with 15% FBS, heparin, bovine
endothelial cell growth factor, and antibiotics as above.

Preparation of L-[35S]Hcy Thiolactone

and L-[35S]Hcy
The method of Baernstein19 was used to convert 5 mCi L-[35S]Met
(Amersham) to L-[35S]Hcy thiolactone. L-[35S]Hcy thiolactone was
purified by 2-dimensional thin-layer chromatography (TLC).8 Fresh
[35S]Hcy was prepared for each experiment by hydrolysis of
L-[35S]Hcy thiolactone (1 mmol/L, 10 000 Ci/mol) with NaOH in the
presence of DTT.8

Preparation of HDL
HDLs were prepared from human serum by ultracentrifugation in
potassium bromide (density of 1.225 g/mL) and gel filtration20 on a
Sephacryl HR S-300 (Pharmacia) column in the presence of
1 mmol/L CaCl2.

lation of DTT-treated samples followed by 1-dimensional TLC on

cellulose plates.8 Determinations of [35S]Hcy and [35S]Met in cellular
and extracellular proteins, treated with 10 mmol/L DTT (5 minutes,
25C), were carried out as described.8 DTT-labile Hcy represented
up to 60% of total Hcy incorporated into protein.

Edman Degradation
Edman degradation of DTT-treated and carboxymethylated
[35S]Hcy-labeled protein was carried out as described by Chang.21
Standard of phenylthiohydantoin (PTH)(S-carboxymethyl)-Hcy
was prepared by Edman degradation of in vitro homocysteinylated
human serum proteins.10

Results
Synthesis of Hcy Thiolactone From
Endogenous Hcy
In human cells, endogenous Hcy is formed from Met in
enzymatic reactions that start with the formation of
S-adenosyl (Ado)Met. As a result of subsequent methylation
reactions, AdoMet is converted into AdoHcy. AdoHcy is then
hydrolyzed to Hcy, which can be converted into thiolactone,8,14 particularly when the conversion to Met through the
folate- and vitamin B12 dependent transmethylation, or to
cysteine through the vitamin B6 dependent trans-sulfuration
pathway, is compromised.2 To examine thiolactone synthesis
from the endogenous Hcy in HUVECs, we incubated the cells
with 5 mol/L [35S]Met for up to 48 hours. Most (95%)
[35S]Met was metabolized within 20 hours. The 3% of
[35S]Met that remained in the media after 20 hours (Table 1)
is due, most likely, to protein turnover. Eighty-three percent
of the metabolized [35S]Met was incorporated into protein as
determined by trichloroacetic acid precipitation of cell ex-

[35S]Met and [35S]Hcy Labeling Conditions

HUVECs were grown to confluence on 3.5-cm dishes. The medium
was then replaced with 0.5 mL of methionine (Met)free M199
supplemented with 15% dialyzed FBS, heparin, and bovine endothelial cell growth factor; 5 mol/L [35S]Met (0.05 mg/mL); or 5 to
100 mol/L [35S]Hcy (0.05 mg/mL), and the cultures were maintained at 37C/5% CO2 for up to 48 hours. Because streptomycin
destroys Hcy thiolactone (see Results), we thoroughly rinsed the
cells with streptomycin-free medium before adding fresh Met-free
and streptomycin-free medium containing 35S-labeled amino acids.
Even though fresh [35S]Hcy was added to the medium at the
beginning of an experiment, it oxidized within 2 to 4 hours to
[35S]Hcy and mixed protein-S[35S]Hcy and Cys-S[35S]Hcy
disulfides.

Determination of Hcy Thiolactone, Hcy,

and Hcy-Protein
Hcy thiolactone was determined by 2-dimensional TLC on cellulose
plates (Kodak).8,11,12 Total Hcy was determined by carboxymethy-

Figure 1. Metabolic conversion of [35S]Met to [35S]Hcy and

[35S]Hcy thiolactone in HUVEC cultures: effect of supplementation with folic acid. Confluent HUVEC cultures were labeled with
5 mol/L [35S]Met (10 000 Ci/mol). Time courses of [35S]Hcy
thiolactone (A) and [35S]Hcy (B) synthesis in the absence ( f )
and presence (F) of 10 mol/L folic acid are shown.

Jakubowski et al

Figure 2. Factors affecting metabolic conversion of [35S]Hcy to

[35S]Hcy thiolactone in HUVEC cultures. Confluent HUVEC cultures were labeled with 10 to 80 mol/L [35S]Hcy (625 to 5000
Ci/mol). A, Time courses of [35S]Hcy thiolactone synthesis from
10 ( f ), 20 (), 40 (F), and 80 ( ) mol/L [35S]Hcy. B and C,
Time courses of [35S]Hcy thiolactone synthesis from 10 (B) and
80 (C) mol/L [35S]Hcy and 0 ( f ), 5 (), 10 (F), and 20
() mol/L Met. D, Time courses of [35S]Hcy thiolactone synthesis from 10 mol/L [35S]Hcy in the absence ( f ) and presence of
10 mol/L folic acid (), 1.5 mol/L methylcobalamine (F), 10
mol/L folic acid1.5 mol/L methylcobalamine ( ), and 1
mg/mL HDL ().

tracts (not shown). About 4% of the metabolized [35S]Met

was converted into [35S]Hcy thiolactone (Figure 1A), most of
which accumulated in the culture medium (Table 1). In
agreement with previous studies,22 13% of [35S]Met was
metabolized to [35S]Hcy, most of which accumulated in the
culture medium (Figure 1B). In addition, folic acid inhibited
thiolactone synthesis in HUVECs, most likely by facilitating
transmethylation of Hcy to Met by Met synthase. This was
shown by the addition of 10 mol/L folic acid to HUVEC
cultures. Such an addition prevented accumulation of both
thiolactone (Figure 1A) and Hcy (Figure 1B) in the culture
medium and in cells (Table 1). These data demonstrate that
endogenous Hcy is efficiently metabolized to thiolactone by
HUVECs and that the metabolism is inhibited by supplementation with folic acid.

Thiolactone and Hcy-Protein in Endothelial Cells

47

Figure 3. Turnover of Hcy thiolactone in HUVEC cultures.

[35S]Hcy thiolactone (5 mol/L) was incubated in M199 supplemented with 15% dialyzed FBS, heparin, and bovine endothelial
cell growth factor in the absence (F) and presence ( ) of confluent cells (A) or in the absence (F) and presence ( ) of 0.1
mg/mL streptomycin (B).

[35S]Hcy, 5 to 20 mol/L Met progressively inhibited the

synthesis of [35S]Hcy thiolactone (Figures 2B and 2C). At
incubation times 20 hours, a greater degree of inhibition by
Met was found than at later times. Even in the presence of
20 mol/L Met, up to 1 mol/L [35S]Hcy thiolactone was
formed from 80 mol/L [35S]Hcy (Figure 2C). With
40 mol/L [35S]Hcy, Met progressively inhibited the synthesis of [35S]Hcy thiolactone, reaching as high as 80% inhibition
at 40 mol/L Met after 48 hours (Figure 4D). These results
suggest involvement of methionyl-tRNA synthetase in thiolactone synthesis in HUVECs.

Supplementation With Folic Acid or HDL Inhibits

Synthesis of Hcy Thiolactone

The kinetics of thiolactone synthesis from exogenous Hcy

were examined at physiological concentrations of Hcy (10 to
80 mol/L), folic acid (26 nmol/L; Reference 22), and
vitamin B12 (180 pmol/L from serum; Reference 22). Thiolactone accumulation positively correlated with the incubation time and Hcy concentration (Figure 2A). Up to 4 mol/L
thiolactone accumulated in culture media under these conditions, representing up to 5% exogenous Hcy. There was very
little (0.1%) incorporation of 35S label from exogenous Hcy
into cellular protein, as determined by trichloroacetic acid
precipitation of cell extracts (not shown). Thus, metabolic
conversion of Hcy to thiolactone represents a major pathway
under these conditions.

Effects of supplementation with folic acid, methylcobalamine, and HDL on kinetics of thiolactone synthesis from
exogenous Hcy in HUVEC cultures are shown in Figure 2D.
At low [35S]Hcy (10 mol/L), supplementation with
10 mol/L folic acid resulted in 90% inhibition of thiolactone synthesis (Figure 2D). At high [35S]Hcy (80 mol/L),
inhibition of thiolactone synthesis by folic acid was less
pronounced (75%), and significant (1 mol/L) thiolactone
levels still formed (not shown). Methylcobalamine
(1.5 mol/L) had only a minor effect (10% to 20% inhibition)
on thiolactone synthesis (Figure 2D). About 5% of 35S label
from exogenous [35S]Hcy was incorporated into cellular
protein from folic acidsupplemented cultures (not shown).
More Met and less Hcy was present in cells maintained in
folic acidsupplemented medium than in unsupplemented
medium (Table 1). This finding supports the role of folate in
transmethylation of Hcy to Met also in HUVECs.
Because it contains tightly bound Hcy thiolactonase as one
of its components,23 HDL should inhibit accumulation of Hcy
thiolactone. Indeed, supplementation of HUVEC cultures
with HDL led to 80% inhibition of thiolactone synthesis
(Figure 2D). The half-life of thiolactone decreased from 3.5
hours in unsupplemented cultures to 10 minutes in HDLsupplemented cultures (not shown).

Met Inhibits Synthesis of Hcy Thiolactone

Turnover of Hcy Thiolactone

We next examined the effects of physiological concentrations

of Met on the kinetics of thiolactone synthesis from [35S]Hcy.
At low (10 mol/L) and high (80 mol/L) concentrations of

In M199 without antibiotics, thiolactone was hydrolyzed to

Hcy with a half-life of 3.5 hours in the absence and presence
of HUVECs (Figure 3A), indicating that the turnover of

Level of Hcy Thiolactone Is Proportional to the

Concentration of Exogenous Hcy

48

Circulation Research

July 7, 2000

Figure 4. Incorporation of Hcy into protein in HUVEC cultures.

Confluent HUVEC cultures were labeled for 48 hours with 3 to
100 mol/L [35S]Hcy (330 to 5000 Ci/mol). Shown are levels of
protein-Hcy ( f ) and protein-Met (F) in intracellular (A) and
extracellular (B) proteins as a function of [35S]Hcy concentration
and levels of Hcy thiolactone as a function of [35S]Hcy (C) and
Met (D) concentration.

thiolactone in HUVEC cultures was due to hydrolysis by the

medium, not by cells. However, in antibiotic-containing
medium, thiolactone was turned over much faster, with a
half-life of 10 minutes (Figure 3B). This was due to a
second-order reaction of thiolactone with streptomycin. The
reaction of thiolactone with streptomycin (k2000 (mol
L)1 h1) was 400 times faster than with lysine (k5 (mol
L)1 h1; Reference 10). Possible chemistry may involve the
aldehyde group of streptomycin forming a Schiff base adduct
with the -amino group of thiolactone.

Incorporation of Hcy Into Protein

To determine whether Hcy can be incorporated into protein,
we prepared cellular and extracellular [35S]proteins from
[35S]Hcy-labeled HUVECs and acid-hydrolyzed them to liberate their [35S]amino acids, which were then separated by
2-dimensional TLC. In addition to [35S]Met, substantial
amounts of [35S]Hcy were present in proteins. The amount of
Hcy incorporated into protein was proportional to Hcy
concentration in HUVEC cultures in the range of 3 to
100 mol/L (Figures 4A and 4B). Hcy incorporation into
TABLE 2.

intracellular proteins ranged from 20% (at low Hcy) to 95%

(at high Hcy) of Met incorporation (Figure 4A). Extracellular
proteins incorporated 2- to 4-fold more Hcy than Met (Figure
4B). Hcy incorporation represented 20% of Hcy thiolactone
present in HUVEC cultures (Figure 4C).
We also examined the effects of supplementation with folic
acid, Met, and HDL, each of which inhibits indirectly or
directly Hcy thiolactone formation, on protein homocysteinylation. Folic acid abolished incorporation of Hcy into
cellular and extracellular protein (Table 2), most likely by
facilitating transmethylation of Hcy to Met, thereby indirectly
preventing synthesis of Hcy thiolactone (Table 1). As expected, 10 times more Met was incorporated into protein in
the presence of 10 mol/L folic acid than in its absence
(Table 2). About 70% inhibition of Hcy incorporation into
protein was observed in the presence of 20 mol/L Met,
possibly because of Met inhibition of thiolactone synthesis
(Figure 4D). HDL, apparently because of its tightly associated Hcy thiolactonase, which hydrolyzes thiolactone23 (Figure 2D), inhibited incorporation of Hcy into extracellular
protein. However, HDL did not affect incorporation of Hcy
into intracellular protein (Table 2). Incorporation of Met into
cellular and extracellular protein was not affected by HDL, as
expected.

Hcy Is Recovered From Proteins Subjected to

Edman Degradation
To determine whether incorporation of Hcy into protein is
post-translational or translational, [35S]proteins from HUVEC
cultures were subjected to Edman degradation. Amino acids
having a free -amino group are released from protein after 1
cycle of Edman degradation. Thus, Hcy incorporated posttranslationally as a result of homocysteinylation by thiolactone of side-chain amino groups of protein lysine residues
would be released by Edman degradation.10
As shown in Figure 5, Edman degradation liberated PTH(S-carboxymethyl)[35S]Hcy from [35S]proteins obtained
from HUVEC cultures incubated with (in mol/L) 10 (lane
1), 40 (lane 2), and 80 (lane 3) [35S]Hcy. Quantification of the
radiolabeled spots by scintillation counting indicated that
PTH-(S-carboxymethyl)[35S]Hcy represented 30% of total
[35S]protein (not shown). Less PTH-(S-carboxymethyl)
[35S]Hcy was liberated from [35S]proteins obtained from
cultures supplemented with 20 mol/L Met (lane 4) or 1
mg/mL HDL (lane 6). Much less PTH-(S-carboxymethyl)
[35S]Hcy was liberated from [35S]proteins obtained from
cultures supplemented with 10 mol/L folic acid (lane 5) or
10 mol/L folic acid and 1.5 mol/L methylcobalamine (lane

Levels of Protein-Hcy and Protein-Met in HUVEC Cultures

Culture Conditions
10 mol/L Hcy, control
10 mol/L folic acid

Intracellular
Protein-Hcy
0.72
0.1

Intracellular
Protein-Met

Extracellular
Protein-Hcy

Extracellular
Protein-Met

105

5.50.3

5.41.7

106

0.1

89

1 mg/mL HDL

0.7

10.1

1.9

6.2

20 mol/L Met

0.25

5.8

1.5

4.8

Data are presented as pmol/105 cells.

Jakubowski et al

Figure 5. Edman degradation of proteins from HUVEC cultures.

Analyses of [35S]proteins from HUVEC cultures incubated with
(in mol/L) [35S]Hcy 10 (lane 1), 40 (lane 2), and 80 (lane 3); 10
[35S]Hcy20 Met (lane 4); 10 [35S]Hcy0 folic acid (lane 5); 10
[35S]Hcy10 folic acid and 1.5 methylcobalamine (lane 7); and
10 [35S]Hcy1 mg/mL HDL (lane 6) are shown. Lane 8, Negative
control with proteins from culture medium (without HUVECs)
incubated with 10 mol/L [35S]Hcy. Lane 9, Standard of PTH-(Scarboxymethyl)-Hcy, indicated by Hcy*. Faint spots of PTH[35S]Met, migrating close to the top of the TLC plate are present
in lanes 5 and 7.

7). Faint spots of PTH-[35S]Met, migrating close to the top of

the TLC plate in lanes 5 and 7, are most likely due to
N-terminal Met that is expected to be observed because of
heavy incorporation of [35S]Met into protein in folic acid
supplemented cultures.

Limited Number of Extracellular Proteins

Become 35S Labeled
To determine whether specific extracellular proteins became
35
S labeled, we subjected to SDS-PAGE cell-free media from
HUVECs maintained on [35S]Hcy, as well as a standard of in
vitro homocysteinylated human serum proteins. As shown in
Figure 6, the SDS-PAGE [35S]protein patterns obtained with
extracellular proteins were different from the pattern of the
standard of human serum N-[35S]Hcyproteins. As suggested
by the prominent band densities and apparent molecular
masses,10 major N-Hcyproteins present in in vitro homocysteinylated serum were albumin (68 kDa), subunits of IgG (50
and 25 kDa), transferrin (80 kDa), and microglobulin (180
kDa) (lane 3 in Figure 6). In addition to the radiolabeled band

Figure 6. SDS-PAGE separation of [35S]proteins from HUVEC

cultures. Extracellular proteins from HUVEC cultures maintained
for 48 hours on (in mol/L) 2 (lane 2), 10 (lane 6), 40 (lane 5),
and 80 (lane 1) [35S]Hcy, and 10 [35S]Hcy10 folic acid (lane 4),
were separated by SDS-PAGE on 10% gels. Specific activity of
[35S]Hcy varied from 10 000 (at low Hcy) to 250 Ci/mol (at high
Hcy). An autoradiogram of a gel is shown.

Thiolactone and Hcy-Protein in Endothelial Cells

49

of albumin, 5 distinct protein bands (of molecular weights

45, 50, and 180, with 2 of 200 kDa) were visible on
SDS-PAGE patterns obtained with protein samples (containing up to 80% [35S]Hcyprotein; see Figure 4B) from
HUVEC cultures maintained on (in mol/L) 2 (lane 2), 10
(lane 6), 40 (lane 5), and 80 (lane 1) [35S]Hcy. These distinct
proteins became more heavily labeled with [35S]Met (Table 2)
in cultures supplemented with folic acid (lane 4). This
suggests that newly synthesized secreted HUVEC proteins,
the identity of which remains to be established, are targets for
protein homocysteinylation.

Discussion
Our data demonstrate that (1) Hcy thiolactone is an important
component of Hcy metabolism in human vascular endothelial
cells; (2) Hcy is incorporated into proteins; and (3) the extent
of thiolactone formation and protein homocysteinylation
depends on the extracellular concentrations of Hcy, folic acid,
and HDL, factors that have been implicated in susceptibility
to vascular disease in humans. Under deregulated metabolic
conditions (low folate, high Hcy), protein homocysteinylation
was increased relative to that under normal metabolic conditions (high folate, low Hcy). The ability to support thiolactone synthesis and protein homocysteinylation may underlie
susceptibility of human endothelial cells to Hcy-induced
damage in atherosclerosis.
Dudman et al17 reported that HUVECs exhibit thiolactonehydrolyzing activity, which we could not find in the present
work. Instead, we detected exceptionally efficient synthesis
of thiolactone in these cells (Figures 1 and 2). A possible
explanation for the finding by Dudman et al17 is that incomplete removal of streptomycin (present in endothelial cell
culture media used by Dudman et al17 ) would lead to a false
thiolactonase activity in an indirect thiol release assay.
However, Lineweaver-Burk plots of the activity observed by
Dudman et al17 make this explanation unlikely. Another
possible explanation for the discrepancy could be differences
between HUVEC donors used in the 2 studies.
HUVECs excrete large amounts of Hcy when they are
cultured in standard Met-containing medium that contains 26
nmol/L folic acid (Reference 22 and this work). However, in
addition to Hcy, HUVECs also synthesized large amounts of
thiolactone. Although 26 nmol/L folic acid is apparently
sufficient to support growth of HUVECs on Met, it is not
sufficient for transmethylation of Hcy to Met in these cells.22
Supplementation of culture media with 10 mol/L folic acid
restored transmethylation of Hcy to Met and prevented
accumulation of both Hcy and thiolactone.
Because Hcy thiolactone forms in the active site of
methionyl-tRNA synthetase,3,5 the synthesis of thiolactone is
expected to increase with an increase in Hcy/Met ratio.25
Indeed, the synthesis of thiolactone by HUVECs increased
with an increase in Hcy concentration and decreased with an
increase in Met concentration. Factors such as folic acid that
affect Hcy/Met ratios in human plasma1,22 also affected
thiolactone synthesis in vascular cells (this work).
Large amounts of Hcy were also found to be incorporated
into cellular and extracellular proteins in HUVEC cultures,
with up to 4-fold more Hcy than Met being incorporated in

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July 7, 2000

the presence of high Hcy and low folate levels. The exact
mechanism of Hcy incorporation into protein is unknown.
However, the recovery of Hcy from these proteins by Edman
degradation suggests that Hcy incorporation is most likely
due to homocysteinylation of protein amino groups by thiolactone.10 Not only thiolactone synthesis but also protein
homocysteinylation was affected by the levels of Hcy, folate,
and HDL, all of which have been linked to vascular
diseases.16
Elevated levels of Hcy are an independent risk factor for
vascular diseases.1 However, it is not clear why excess Hcy is
harmful. The following hypotheses have been proposed to
explain the effects of Hcy. Hcy may induce oxidative damage
of endothelial cells,24 promote vascular smooth muscle
growth, and inhibit regeneration of endothelial cells.25 Inhibition of endothelial cells growth may result from inhibition
of methylation by Hcy.25 In addition, Hcy may affect the
blood-clotting mechanisms, thereby enhancing a prothrombotic state.26 Alterations in the expression of multiple genes
induced by Hcy in endothelial cells could also contribute to
atherosclerosis.27 However, in most experiments that led to
these hypotheses, nonphysiological concentrations of Hcy (1
to 10 mmol/L) were used, and, where reported, similar effects
were observed with cysteine or 2-mercaptoethanol.24,26,27
Although effects of Hcy on growth and methylation were
found at physiological Hcy concentrations, these effects
could only be observed in the presence of high levels of
adenosine,25 which are not present in human cells.
Our findings that thiolactone and Hcy-protein are present
in HUVEC cultures support an alternative hypothesis,
namely, that metabolic conversion of Hcy to thiolactone,
protein homocysteinylation, and resulting protein damage10
cause Hcy toxicity to human endothelium. Protein homocysteinylation is specific for Hcy because thiolactonethe
metabolite generating the modification can only arise from
Hcy.5,8 14 The synthesis of thiolactone and protein homocysteinylation occur at physiological concentrations of Hcy and
depend on the Hcy/Met ratio, which in turn depends on folate.
By using folate-limited media, we were able to obtain the
levels of thiolactone and protein-Hcy in HUVEC cultures at
least 100 times higher than the levels observed in previous
experiments with fibroblast cultures,8 thus demonstrating that
the lack of folate is a major determinant of efficient thiolactone synthesis and protein homocysteinylation.
Hcy thiolactone is known to be toxic to endothelial cells.
For example, chronic infusions of baboons with Hcy thiolactone cause endothelial cell injury.28 Thiolactone, but not Hcy,
was found to induce gross changes in endothelial cell morphology and to induce cell death.29 It should be noted,
however, that it is not known whether these effects were due
to protein homocysteinylation.
That protein homocysteinylation may be physiologically
detrimental is suggested by several studies. For instance,
enzymes, such as MetRS and trypsin,10 are inactivated by
homocysteinylation. Lysyl oxidase, an important enzyme
responsible for post-translational modification essential for
the biogenesis of connective tissue matrices, is inactivated by
Hcy thiolactone, which modifies the active-site tyrosinequinone cofactor.30 In addition, homocysteinylated cytochrome c

is particularly prone to aggregation,10 and protein homocysteinylation can be physiologically detrimental by eliciting
immune response, as shown in rabbits immunized with
homocysteinylated LDL.31
In conclusion, this work shows that in human vascular
endothelial cells Hcy is incorporated into proteins and that
this incorporation, as well as thiolactone formation, is dependent on extracellular Hcy, folic acid, and HDL. At this stage,
our findings and observations by others, suggesting that
protein homocysteinylation impairs physiological function,
do not establish causality of Hcy in atherosclerosis. However,
they underscore the importance of examining protein homocysteinylation in the context of Hcy-induced pathologies in
humans.

Acknowledgments
This research was supported in part by grants from the National
Science Foundation (MCB-972-4929 to H.J.) and the NIH (HL47906
to A.A.).

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