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133) Associated with Extreme Obesity Undergoes Proteasomal Degradation after Defective Intracellular Transport1
Departments of Medicine (H.R., S.O.R., J.P.W.) and Clinical Biochemistry (H.R., B.J.R., S.O.R., J.P.W.), University of Cambridge, Addenbrooke’s Hospital, Cambridge, CB2 2QR, United Kingdom
Address all correspondence and requests for reprints to: Prof. Stephen O’Rahilly, Departments of Medicine and Clinical Biochemistry, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom. E-mail: sorahill{at}hgmp.mrc.ac.uk
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Abstract |
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133) mutant leptin. In the present investigation, we have
examined the mechanisms underlying the defective secretion of the
133 leptin in transient transfection studies in Chinese hamster
ovary and monkey kidney epithelium cells. Consistent with our
previous observations, only immunoreactive wild-type (wt) leptin was
secreted. In pulse chase experiments, intracellular wt leptin levels
decreased, concomitant with secretion into the medium. In contrast,
though immunoreactive 133 leptin disappeared from cell lysates with
kinetics similar to those of wt leptin (half-life, 45 min), it
was not detected in the medium. Inhibition of the proteasome, using the
inhibitor clastolactacystin ß-lactone, led to a significant increase
in the intracellular levels of 133 leptin, indicating a role for the
proteasome in the degradation pathway. Although intracellular
immunoprecipitated wt and 133 leptin levels were comparable,
analysis of total cell lysates revealed a 7-fold increase in total
intracellular 133 leptin, compared with wt leptin. Size-exclusion
membrane filtration demonstrated that intracellular 133 leptin
accumulated in an aggregated form, presumably as a result of misfolding
in the endoplasmic reticulum. Consistent with this, an endoplasmic
reticulum-like localization for 133 leptin was detected by
immunofluorescence microscopy. In conclusion, the 133 mutant leptin
is not secreted but accumulates intracellularly, as a consequence of
misfolding/aggregation, and is subsequently degraded by the proteasome.
These studies further define the genotype/phenotype correlation in this
paradigmatic case of human leptin deficiency. |
Introduction |
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Previously, we reported a homozygous mutation in the leptin gene in 2
extremely obese children with undetectable serum leptin (1). The
deletion of a single guanine nucleotide in codon 133 results in a
frameshift mutation that leads to the introduction of 14 aberrant amino
acids, followed by a premature stop codon. Preliminary data suggested a
defect in the secretion of this truncated (133) mutant leptin (1).
More recently, a homozygous missense mutation (R105W) in the human
leptin (ob) gene was also found to associate with
undetectable serum leptin, extreme obesity, and impaired secretion in
transfected cells (8).
Wild-type (wt) leptin contains an intrachain disulphide bond (9)
that seems necessary for its biological activity (10). The 133
leptin protein lacks this disulphide bond, suggesting that, even if it
were secreted, the protein would be inactive. In contrast, the
intrachain disulphide bond may be intact in the R105W leptin; thus, it
is less clear whether the R105W leptin protein would exhibit biological
activity if its secretion defect were overcome. Wt leptin does not seem
to be stored in adipocytes or in other cultured cell lines, suggesting
that its secretion is constitutive rather than regulated. As a result,
leptin secretion is thought to be regulated only by changes in
transcription and/or translation (11, 12, 13).
We have investigated the intracellular fate of the first reported
functionally significant mutant human leptin protein, 133, and its
effect on synthesis and secretion of wt leptin. We demonstrate, by
transfection studies in Chinese hamster ovary (CHO) and monkey kidney
epithelium (COS7) cells, that the nucleotide deletion at codon 133
disrupts the intracellular trafficking and secretion of the truncated
leptin protein. The proteasome is the major cytosolic protease
responsible for the degradation of misfolded proteins, which are
usually secreted or expressed at the cell surface (see Ref. 17). By use
of the proteasome inhibitor, clastolactacystin ß-lactone, we
implicate the proteasome in the degradation pathway of the truncated
leptin protein. Cotransfection studies indicated that mutant leptin did
not interfere with the synthesis and secretion of wt leptin. We have
also confirmed the secretion defect reported for the R105W leptin in a
second cell type. Attempts to overcome the secretion defect of the
mutant leptins were without effect.
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Materials and Methods |
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Cell lines. CHO.K1 cells were grown in Nutrient F-12 HAM medium supplemented with 10% FBS, 105 U/liter penicillin, 0.1 g/liter streptomycin, and 2 mM L-glutamine. COS7 cells were cultured in DMEM (4.5 g/liter glucose) supplemented as above. Cells were grown at 37 C under an atmosphere of 95% air-5% CO2 unless otherwise stated. Cells grown at 29 C were incubated in HEPES-buffered Basal Medium Eagle medium.
Construction of R105W mutant leptin. All routine DNA
manipulations were performed using standard protocols (14). The pRc.CMV
expression vectors (Invitrogen, Leek, Netherlands),
encoding full-length wt or 133 human leptin, have been described
previously (1). The R105W mutant leptin complementary DNA was
constructed using a QuikChange Site-Directed Mutagenesis Kit
(Stratagene, Cambridge, UK), in accordance with the
manufacturer’s instructions, and confirmed by direct sequencing.
Transient transfection of cells. Transfections were
performed on cells grown in 30-cm2 wells using equal
amounts of the pRc.CMV expression vector encoding full-length wt, or
133, or R105W mutant human leptins and Lipofectamine (Gibco BRL, Paisley, UK), in accordance with the manufacturer’s
instructions. Experiments were performed approximately 24 h after
transfection.
Immunoprecipitation and Western blotting. Immunoprecipitation and Western blotting were performed as described previously (15). In brief, for determination of secreted leptin, cells were maintained in fresh medium for approximately 20 h before analysis. Cultured medium was collected on ice, and an equal volume of lysis buffer (2x) was added before immunoprecipitation. For determination of intracellular leptin, cells were rinsed once in ice-cold PBS, lysed, harvested, and precleared by centrifugation. Leptin was immunoprecipitated from samples using antileptin antibody (diluted 1:100) and 50 µl of a 5% slurry of protein A-agarose. Immunoprecipitates, or total cell lysates, were electrophoresed on 15% SDS-polyacrylamide gels (SDS-PAGE) and were transferred by electroblotting to Immobilon-P polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA). Blots were probed with antileptin antibody (diluted 1:5000), followed by [125I]-labeled goat antirabbit antibody (16). Proteins were visualized using a Fujix BAS 2000 PhosphorImager, Tokyo, Japan.
Metabolic labeling studies. Transfected cells were preincubated in prewarmed methionine and cysteine-free DMEM for 1 h. Then, 100 µCi [35S] EasyTag Express Methionine/Cysteine Protein Labeling Mix (DuPont NEN, Hertfordshire, UK) was added to each well, and cells were further incubated for 45 min. Cells were then washed once using complete HAMS F12 medium and incubated in the same. At the appropriate time points, media and cells were harvested, and leptin was immunoprecipitated as described above. Immunoprecipitates were electrophoresed on 15% SDS-PAG and analyzed using a Fujix Bas 2000 PhosphorImager.
Proteasome inhibition. Transfected cells were untreated, treated with vehicle alone [dimethyl sulfoxide (DMSO) at a final concentration of 0.25%], or treated with vehicle containing the irreversible proteasome inhibitor clastolactacystin ß-lactone (Calbiochem-Novabiochem, San Diego, CA) at a final concentration of 10 µM for 2 h or 16 h. Total cell lysates were analyzed by Western blotting, as described.
Immunofluorescence microscopy. CHO cells were transfected using Tfx10, according to the manufacturer’s instructions (Promega Corp., Southampton, UK). The proteasome was inhibited by lactacystin, as described above. Cells were fixed with 4% paraformaldehyde, permeabilized with methanol, blocked with 0.2% BSA, incubated first with the antileptin antibody, PCB (1:100), and then with goat antirabbit Texas Red-linked Ig (1:100, Jackson ImmunoResearch, Baltimore). Immunofluorescence microscopy was performed using a Bio-Rad MRC 1000 apparatus (Bio-Rad Laboratories Inc., Hertfordshire, UK).
Size-exclusion membrane filtration columns. Cell lysates from transfected CHO cells were run over Microcon-100 microconcentrators (Amicon, Beverly, MA), according to the manufacturer’s instructions. Proteins collected within eluates and retentates were precipitated using trichloroacetic acid and analyzed by Western blotting, as described.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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133 human leptin, intracellular and secreted
leptin levels were determined by immunoprecipitation and Western
blotting (Fig. 1
). Though intracellular
levels of immunoreactive wt (16 kDa) and 133 (14 kDa) leptin were
comparable, only immunoreactive wt leptin was detected in the cultured
medium from transfected CHO and COS7 cells. When cells were
cotransfected with both wt and 133 leptin, again both wt and 133
leptin were found in cell lysates, and only immunoreactive wt leptin
was found in the medium (Fig. 1). Thus, 133 leptin did not seem to
be secreted from cells. Expression of 133 leptin did not inhibit the
secretion of wt leptin from the same cells, because wt leptin was
secreted with similar efficiency in the absence or presence of mutant
leptin (100% vs. 92 ± 18%, respectively, leptin
secreted per microgram of wt complementary DNA).
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133 leptin, pulse chase
studies were performed. Transfected CHO cells were metabolically
labeled for 45 min and then chased for up to 6 h in complete
medium. Leptin was then immunoprecipitated from cell lysates and
medium, subjected to SDS-PAGE, and analyzed using a PhosphorImager. The
levels of intracellular radiolabeled wt leptin decreased with a
concomitant increase in secreted leptin (Fig. 2, a and b). Although the time course of
the disappearance of intracellular radiolabeled 133 leptin was
similar to that of wt leptin, with both exhibiting a half-life
of approximately 45 min, no 133 leptin was detected in the
medium.
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133 leptin, we were also unable to detect
accumulation of 133 leptin within the cells. There are several
possible explanations for these observations. The 133 leptin may
become misfolded or aggregated, such that it is no longer
immunoreactive; or it may be recognized as being improperly folded, and
so, targeted for rapid degradation. To elucidate the fate of the 133
leptin, we first examined the levels of leptin in cell lysates without
immunoprecipitation. In contrast to the comparable levels of
intracellular wt and 133 leptin seen after immunoprecipitation of
samples, analysis of samples without immunoprecipitation revealed there
to be approximately 7-fold more intracellular 133 leptin, compared
with wt leptin (Fig. 3A, lanes a and d).
Indeed, the efficiency of immunoprecipitation of 133 leptin was
found to be markedly lower than that of wt leptin [10–15%, compared
with 80–90%, respectively (Fig. 3A, lanes b and e; and Fig. 3B).
Consistent with the observation that 133 leptin was
immunoprecipitated less efficiently than wt leptin, significant amounts
of 133 leptin remained in the supernatants of immunoprecipitates;
but this was not the case for wt leptin (Fig. 3A, lanes c and f; and
Fig. 3B). These findings were confirmed using three different
polyclonal antileptin antibodies (data not shown) and suggest that the
133 leptin exists in a conformation that differs markedly from wt
leptin.
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133
leptin was not secreted, but accumulated within the cells, we examined
whether the proteasome played a role in degradation of the mutant
leptin. Transfected CHO cells were treated with the proteasome
inhibitor, clastolactacystin ß-lactone, at a final concentration of
10 µM, such that greater than 95% of proteasome activity
would be irreversibly inhibited (18, 19). Incubation of cells, in the
presence of the proteasome inhibitor for 2 h, had no clear effect
(data not shown); however, treatment for 16 h resulted in an
increase of approximately 8-fold in the amount of intracellular 133
leptin (Fig. 4). Intracellular wt leptin
levels were also slightly increased after 16 h of incubation with
the proteasome inhibitor, but to a much lesser degree (
2-fold) than
that seen for mutant leptin (Fig. 4). We believe that this modest
effect on wt leptin is attributable to the overexpression of such an
exogenous protein in CHO cells.
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133 leptin in transfected
cells by immunofluorescence microscopy were unsuccessful, although a
cytoplasmic marker protein, which was cotransfected, was readily
detected in approximately 10–20% of cells (data not shown). We
reasoned that inhibition of the proteasome, which results in an
increase in intracellular levels of 133 leptin, in particular, may
favor visualization of intracellular leptin. Such proteasomal
inhibition did not facilitate detection of wt leptin in transfected
cells by immunofluorescence microscopy (data not shown). However, upon
treatment of cells with the proteasome inhibitor, we were able to
detect intracellular 133 leptin in a staining pattern consistent
with localization in the endoplasmic reticulum (Fig. 5).
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133 leptin accumulated as aggregates within
cells, total cell lysates were applied to size-exclusion membrane
filtration columns with a theoretical cut-off of approximately 100 kDa,
and the eluates and retentates were analyzed by immunoblotting (Fig. 6). These columns allow greater than 95%
of proteins of less than 25-kDa molecular mass to flow through in the
eluate. As expected, wt leptin was detected in the eluate, but the
133 leptin was found only in the retentate. The absence of 133
leptin in the eluate was consistent with the 133 leptin aggregating
within the cell. Interactions with other proteins, such as chaperones,
may also be expected to prevent flow through to the eluate and cannot
be ruled out. That said, we were unable to detect increased
association/retention of the chaperone Ig heavy-chain binding protein
with 133 leptin, compared with wt leptin, in the above studies (data
not shown), even though this chaperone is reported to show increased
interactions with misfolded proteins (20).
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133 leptin was also not detected in cultured medium when analyzed by
direct immunoblotting or after application to size-exclusion membrane
filtration columns or TCA precipitation (data not shown). This rules
out the possibility that the 133 leptin is secreted in a native form
that is nonimmunoreactive, such as the misfolded/aggregated form, and
is consistent with the 133 leptin being defective in its
secretion.
Defective secretion of the recently described R105W mutant leptin has
been reported in transfected COS cells (8). Consistent with this, we
found that it also exhibited defective secretion in transfected CHO
cells (Fig. 7), with R105W leptin being
undetectable in cultured media, whereas wt leptin was readily detected
(Fig. 7, a and b). Analogous to the accumulation of intracellular
133 leptin, described above, the R105W leptin accumulated
intracellularly, compared with wt leptin (Fig. 7, a and b). Treatment
of cells with low-molecular-weight compounds or incubation at reduced
temperatures has been shown to alleviate folding defects of certain
mutant proteins (21). Similar attempts to relieve the block on R105W
leptin secretion, by incubating cells expressing R105W leptin at 29 C
or in the presence of 6% glycerol, failed to do so (Fig. 7, c and d).
Identical treatment of cells expressing the 133 leptin also had no
effect (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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133 leptin failed to be
secreted from transfected CHO cells (1).
Because these probands represent the first naturally occurring
mutations in human leptin to be described and, in addition, one of the
very small number of human subjects with naturally occurring mutations
in a secreted peptide hormone, we wished to establish the molecular
mechanism underlying defective secretion of the 133 leptin. In the
studies reported above, we have: confirmed that the 133 leptin fails
to be secreted into the culture medium in two independent cell types;
determined that the 133 leptin accumulates within cells; shown that
the 133 leptin is less immunoreactive than wt leptin, presumably as
a result of conformational changes; shown that the 133 leptin forms
macromolecular aggregates within the cell; demonstrated, by using
specific inhibitors of proteasome action, that 133 leptin is
targeted to and degraded by the proteasome; and finally, in
cotransfection experiments, that the 133 leptin does not interfere
with secretion of wt leptin.
Thus, this genetic form of obesity in the two related probands may be added to a growing list of human diseases that are associated with mutations that interfere with protein folding and trafficking. For example, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to retention of the encoded protein within the endoplasmic reticulum and degradation by the ubiquitin-proteasome pathway (17, 22, 23). Other examples of mutations affecting protein trafficking include mutations in the insulin receptor (24, 25) and thyroglobulin (26).
The data we present is consistent with the 133 leptin aggregating in
the endoplasmic reticulum. Accordingly, we were able to demonstrate an
endoplasmic reticulum-like staining pattern for 133 leptin, by
immunofluorescence microscopy, after treatment of the cells with the
proteasome inhibitor. However, we were unable to detect wt leptin in
transfected cells, with or without inhibition of the proteasome,
despite using several polyclonal antibodies against leptin. This is in
contrast to the findings of Barr et al. (27), who were able
to detect intracellular wt leptin in the endoplasmic reticulum of cells
from rat epididymal fat pads, by similar methods. The reason(s) for our
inability to detect wt leptin using these methodologies is unclear.
However, explanations, including low efficiency of translation in
nonadipose cell types and/or rapid transit through the cell, may be
invoked.
Recently, further genetic evidence has come to light confirming the importance of leptin in the control of human body weight. Clement et al. (28) have reported a Kabilian family where severely obese individuals are homozygous for a nonsense mutation in the leptin receptor. A Turkish family has also been reported, in which homozygosity for a missense mutation in the leptin gene (R105W) is associated with extreme early onset obesity and low plasma leptin levels (8). Additionally, the affected adult of this kindred also showed failure of pubertal development. The R105W mutant leptin has been shown to exhibit defective secretion in transient transfected COS cells (8). We have extended this observation to transfected CHO cells. These results indicate that impaired intracellular trafficking of R105W leptin is the likely molecular mechanism underlying the hypoleptinemia and severe obesity described. No detailed studies of the cellular itinerary of R105W mutation have, as yet, been presented.
In both families with mutations in leptin thus far described,
heterozygote family members seem not to be markedly clinically affected
and have leptin levels within the range predicted on the basis of their
body mass index. This suggests that the mutant leptins are unlikely to
interfere with the secretion of wt leptin, and we have confirmed this
in the case of the 133 mutation. The mechanism whereby the single
remaining allele up-regulates its expression to maintain normal leptin
levels and normal body weight remains an area for future
investigation.
It is possible that understanding of the precise cell biological
defects associated with mutant forms of leptin may lead to therapeutic
advances. Various low-molecular-weight compounds (glycerol,
trimethylamine-N oxide or deuterated water), which are known to
stabilize proteins in their native conformation, are effective in
correcting the temperature-sensitive protein-folding defect associated
with the F508 CFTR protein (21). This is also true for mutants of
the tumor-suppressor protein p53, the viral oncogene protein pp60src,
and the ubiquitin-activating enzyme E1 (21).
We therefore reasoned that such treatment of cells expressing the R105W
leptin might assuage the block on secretion. However, incubation of
cells, either with glycerol or at reduced temperature, did not
facilitate secretion of the R105W leptin. The finding that incubation
at reduced temperature did not rescue secretion of the R105W leptin
rules out the possibility that the R105W leptin is a
temperature-sensitive mutant. Whether this mutation also affects leptin
binding has not been tested. As expected, similar treatment of cells
expressing the 133 leptin had no effect on secretion.
In summary, we have more precisely defined the molecular basis for defective leptin secretion in subjects with congenital leptin deficiency. These findings add to our understanding of the relationship between genotype and phenotype in this novel clinical syndrome.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Biology and Biochemistry, University
of Bath, United Kingdom BA2 7AY.
Received August 20, 1998.
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References |
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