| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 9, 2001 as doi:10.1096/fj.00-0727fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-crystallin and decreases the post-translational protein modifications induced by oxidative stress1
* Institute of Protein Biochemistry and Enzymology, C.N.R., Arco Felice, and Department of Experimental Oncology, National Cancer Institute, Naples, Italy;
Institute of Clinical Surgery, 2nd University of Naples, School of Medicine Naples, Italy;
2nd Division of Neurology, 2nd University of Naples, School of Medicine Naples, Italy; and
Scientific Department, Sigma Tau S.p.A., Rome, Italy
2Correspondence: Institute of Protein Biochemistry and Enzymology (IBPE), Via Toiano 6, 80072 Arco Felice, Naples, Italy. E-mail: peluso{at}dafne.ibpe.na.cnr.it
SPECIFIC AIMS
Oxidizing free radicals reduce the chaperone activity of lens
-crystallin and increase the susceptibility of lens proteins to
serve as substrate for transglutaminase (TGase) activity, thereby
leading to the protein cross-linking and cataractogenesis typical of
aging and diabetes. To evaluate whether L-carnitine protects
-crystallin function in its ability to prevent aberrant protein
associations and inhibits TGase-induced protein cross-linking, we
measured the molecular chaperone activity of -crystallin and
isopeptide cross-links in rat lenses exposed in vitro to
H2O2 in the presence and
absence of L-carnitine and in control (untreated) lenses.
PRINCIPAL FINDINGS
1. L-carnitine prevents depletion of free carnitine but not of
reduced glutathione (GSH), increases acetyl-L carnitine concentration,
and preserves cell integrity in lens stressed with
H2O2
Concentrations of free carnitine and GSH (which is highly
represented in lens tissue and protects from in vitro diabetic
cataract) were unchanged in control lenses, whereas 500 µM
H2O2 caused a precipitous
drop in levels of free carnitine (156±3 vs. 27±2 µmol/g w-w; in
control and H2O2-treated
lens, respectively P<0.001) and GSH (4.87±0.23 vs.
2.44±0.69 µmol/g w-w; P<0.001). Pretreatment with
L-carnitine (300 µM) did not prevent the
H2O2-induced GSH loss; it
did, however, prevent free carnitine depletion (156±3 vs. 151±2
µmol/g w-w in control and pretreated lenses, respectively). The
H2O2-induced decrease in
free carnitine and GSH paralleled a significant increase of lactate
dehydrogenase (LDH), an indicator of cell integrity, in the medium.
Consequently, depletion of these factors was associated with lens
damage. In lenses pretreated with L-carnitine, the GSH concentrations
approximated those in lenses incubated with
H2O2 alone, but the LDH
concentration in the medium approximated that observed with control
lenses. Pretreatment with L-carnitine not only normalized free
carnitine levels in stressed lenses, but significantly increased the
amount of acetyl-L carnitine, an L-carnitine derivative (29±1 vs.
37±2 nmol/g w-w in control and pretreated lenses, respectively;
P<0.005).
2. L-carnitine prevents the TGase-mediated formation of
water-insoluble protein deposits in
H2O2-treated lenses
Water-insoluble proteins, which constituted only 5% of total
proteins in control lenses, increased to 41% in
H2O2-treated lenses.
Pretreatment with L-carnitine prevented this increase. To determine
whether TGase activity mediated the formation of water-insoluble
protein deposits, we examined total lysates of lens specimens
using Western blotting and a highly specific
anti-
-(
-glutamyl) lysine isopeptide antibody. Very high
molecular mass proteins were prominent in
H2O2-treated lenses; only
minor immunoreactive products appeared in control and
L-carnitine-pretreated lenses (Fig. 1A
).
|
There were 1.3 cross-link residues/100 residues in the high molecular
mass proteins eluted from the gel (see lane 4, Fig. 1A
) and
enzymatically digested. In addition, total lens extract (water-soluble
and -insoluble proteins) from control lenses contained 1–2 cross-link
residues vs. 9–12 cross-link residues/10,000 residues in
H2O2-treated lenses. The
latter value coincides with the insoluble protein concentration in
H2O2-treated lenses.
Therefore, the greater number of cross-links in these lenses originate
from insoluble proteins. Cross-links in lenses pretreated with
L-carnitine overlapped the control value (Fig. 1A
, lanes 2
and 3). The proteolytic digest of water-insoluble lens proteins showed
a chromatographic peak corresponding to the isopeptide only in lenses
treated with H2O2 alone
(Fig. 1B
).
3. L-carnitine protects the -crystallin chaperone activity that
prevents the protein aggregation
protein
cross-linkingcaractogenesis cascade
The chaperone properties of purified water-soluble -crystallin
were determined by the ßL-crystallin (target
protein) aggregation assay. Characteristically,
ßL-crystallin aggregates at elevated
temperatures. -Crystallin from control lenses inhibited the
heat-induced aggregation of ßL-crystallin.
After H2O2, the capacity of
-crystallin to prevent the heat-induced aggregation of
ßL-crystallin significantly decreased;
pretreatment with L-carnitine prevented this negative effect.
Since intermediate filaments such as porcine glial fibrillary
acidic protein (GFAP) are a physiological target of -crystallins, we
tested -crystallin chaperone function using falling ball
viscosimetry in the gel forming assay. By exerting chaperone activity,
-crystallin disaggregates GFAP cytoplasmic inclusions. In the
absence of -crystallin, GFAP forms a protein gel that supports a
ball. -Crystallin from control lenses prevented GFAP gel formation,
whereas -crystallin from
H2O2-treated lenses did
not. -Crystallin from lenses pretreated with L-carnitine blocked
GFAP gel formation to the same extent as -crystallin from control
lenses.
CONCLUSIONS
This study shows that exposure of lenses to
H2O2 significantly reduces
free carnitine content, decreases the solubility of lens proteins, and
alters -crystallin’s chaperone activity. Free carnitine and GSH
levels in control lenses approximated those in fresh lenses.
Consequently, the organ culture conditions, at least those of the
24 h cultures, mimic the lens physiological environment although
differences in the O2 concentrations of organ
culture and in vivo conditions cannot be ruled out.
Lenses treated with L-carnitine withstood oxidative stress. L-carnitine per se is not known to exert antioxidant activity, at least under the conditions used in this study. It did not prevent GSH depletion, which means that the beneficial effect was not mediated by an increase of GSH through, for example, an anaplerotic effect on NADPH, a cofactor of glutathione reductase. The fact that L-carnitine prevented oxidative stress-induced LDH leakage into the medium indicates that it sustains lens integrity. This effect may be related at least in part to the capacity of carnitine and its acyl esters to repair the membrane phospholipids that are damaged by oxidative insult.
We also demonstrate that in vitro oxidative stress diminishes
lens -crystallin chaperone activity and provide evidence that lens
proteins subjected to oxidative insult sustain a high degree of
posttranslational modifications. L-carnitine protected the chaperone
activity of -crystallin and reduced the increased posttranslational
modifications of lens proteins. L-carnitine could contribute to
acetylation of the protein, a process that seems to protect crystallin
from molecular modifications that decrease its chaperone activity. The
relatively large pool of lens carnitine acts as a buffering system that
stabilizes the ratio of acetylated to free coenzyme A. Free carnitine
can be acetylated whenever a mismatch occurs between the fluxes through
pyruvate dehydrogenase and the tricarboxylic acid cycle, as occurs in
cell oxidative stress. In fact, acetyl-L-carnitine was increased in
lens exposed to H2O2 in the
presence of L-carnitine.
Alpha-crystallins are amino-terminally acetylated, and the
N-acetylated-terminal methionine of this crystallin can be oxidized to
methionine sulfoxide in vivo. Oxidation of the amino-terminal
methionine, which is exposed on the surface of the polypeptide, can
negatively affect protein function. Also the, -amino groups of
lysine (Lys) residues are subject to acetylation. All seven Lys
residues of bovine -A-crystallin react with aspirin, the extent of
acetylation varying from 10% for Lys 88 to 60% for Lys166. Aspirin, a
putative anticataract agent, inhibits both glycation and carbamoylation
as well as lens protein aggregation, presumably through acetylation of
Lys residues. Through acetylation of the potential glycation sites,
acetyl-L-carnitine inhibits glycation of -crystallin to a greater
extent than do other crystallins. Only glycation products are involved
in protein cross-linking and in a significant decrease of the
-crystallin chaperone activity.
It remains to be established whether or not L-carnitine is involved in maintaining correct crystallin folding in cells exposed to oxidative stress. We have proposed that as a member of the methylamine family, carnitine be viewed as an organic compatible solute that stabilizes protein, thus mimicking the ions of the Hofmeister series. In the Hofmeister series, the degree of methylation of the nitrogen atoms of substituted ammonium ions enhances their ability to stabilize macromolecules. Thus, in the case of organic methylamine, it is not inconceivable that fully methylated compounds such as carnitine could be one of the most stabilizing factors.
Under our conditions, the TGase-induced increase of lens protein
cross-links was associated with oxidative stress and a decrease in
-crystallin chaperone activity. Why does oxidative stress increase
the degree of TGase-mediated modifications in the lens? Oxidation may
induce conformational changes in lens crystallins that unmask specific
domains, thereby increasing their susceptibility to act as TGase
substrates. Alternatively, the increased susceptibility to TGase might
result from a decrease in -crystallin chaperon activity to levels
where it no longer prevents aggregation of lens crystallins. Changes in
the stability of lens proteins could be crucial in regulating the
posttranslational modifications that occur during cataract development,
akin to the reversible self-assembly of fibrin molecules into a clot
that greatly accelerates the rate of the TGase catalyzed cross-linking
reaction.
It is not known when and how TGase is activated in the lens. The GTP concentration could affect its cross-linking potential, and a significant drop in GTP concentration, as occurs in cataract formation, would significantly favor TGase expression. L-carnitine depletion could lead to decreased energy production from mitochondrial oxidation and consequently to reduced ATP and GTP synthesis, which in turn would result in increased TGase activity. The protective effect of L-carnitine on TGase-mediated cross-link formation could be related to the molecule’s capacity to favor acetylation of target proteins. As occurs during glycation, lysine residues participate in the TGase-mediated cross-linking reaction whereas the acetylated form is no longer a substrate of the enzyme.
It is likely that a combination of two or more of
the above-mentioned mechanisms (see Fig. 2
) underlie the protective effect exerted by L-carnitine on lens
transparency.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0727fje ; to cite this
article, use FASEB J. (May 9, 2001) 10.1096/fj.00-0727fje 
This article has been cited by other articles:
|
|
 |
|
  L. Cerchia, A. D'Alessio, G. Amabile, F. Duconge, C. Pestourie, B. Tavitian, D. Libri, and V. de Franciscis An Autocrine Loop Involving Ret and Glial Cell-Derived Neurotrophic Factor Mediates Retinoic Acid-Induced Neuroblastoma Cell Differentiation Mol. Cancer Res., July 1, 2006; 4(7): 481 - 488. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E Pascale, E Battiloro, G C. Reale, R Pietrobono, M G Pomponi, P Chiurazzi, R Nicolai, M Calvani, G Neri, and E D'Ambrosio Modulation of methylation in the FMR1 promoter region after long term treatment with L-carnitine and acetyl-L-carnitine J. Med. Genet., June 1, 2003; 40(6): e76 - 76. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
