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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0954
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 11 4494-4499
Copyright © 2008 by The Endocrine Society

Mutations in the Amino-Terminal Region of Proopiomelanocortin (POMC) in Patients with Early-Onset Obesity Impair POMC Sorting to the Regulated Secretory Pathway

John W. M. Creemers1, Yung Seng Lee1, Robert L. Oliver1, Mithat Bahceci, Alpaslan Tuzcu, Deniz Gokalp, Julia Keogh, Stefan Herber, Anne White, Stephen O'Rahilly and I. Sadaf Farooqi

University of Leuven (J.W.M.C.), Center for Human Genetics, B-3000 Leuven, Belgium; University of Cambridge (Y.S.L., J.K., S.O., I.S.F.), Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom; Department of Paediatrics (Y.S.L.), Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117595; University of Manchester (R.L.O., A.W.), Endocrine Sciences Research Group, Faculties of Life Sciences and Medical and Human Sciences, Manchester M13 9PL, United Kingdom; Department of Endocrinology and Metabolism (M.B., A.T., D.G.), Dicle University School of Medicine, 21280 Diyarbakir, Turkey; and Princess of Wales Hospital (S.H.), Grimsby DN33 2BA, United Kingdom

Address all correspondence and requests for reprints to: John W. M. Creemers, University of Leuven, Center for Human Genetics, B-3000 Leuven, Belgium. E-mail: John.Creemers{at}med.kuleuven.be.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Mutations in the proopiomelanocortin (POMC) gene that impair the synthesis or structure of POMC-derived peptides predispose to human obesity.

Objective: Our objective was to identify and characterize novel mutations in the POMC gene found in patients with early-onset obesity.

Design and Patients: The POMC gene was screened in 500 patients with severe early-onset obesity. The biosynthesis, processing, sorting, and secretion of wild-type POMC and two newly identified POMC mutants was studied using metabolic labeling, Western blotting, and immunoassay analysis of lysates and conditioned media of transiently transfected β-TC3 cells.

Results: Two novel heterozygous missense mutations in POMC (C28F and L37F) were identified in unrelated probands with early-onset obesity and their overweight or obese family members. Both mutations lie in a region of the N terminus of POMC that has been suggested to be involved in its sorting to the regulated secretory pathway. Metabolic labeling studies indicate that whereas the mutations do not reduce intracellular levels of POMC, both mutations (C28F>L37F) impair the ability of POMC to be processed to generate bioactive products. Studies of the secretion of POMC products suggest, particularly with C28F, that the impaired propeptide processing of these mutations results, at least in part, from a mistargeting of mutant POMC to the constitutive rather than the regulated secretory pathway.

Conclusion: These mutations in patients with early-onset obesity represent a novel molecular mechanism of human POMC deficiency whereby naturally occurring mutations in its N-terminal sequence impair the ability of POMC to enter the trafficking pathway in which serial propeptide processing normally occurs.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Homozygous nonsense or missense mutations in POMC are associated with hyperphagia, early-onset obesity, hypopigmentation, and isolated ACTH deficiency (1, 2, 3). The higher prevalence of obesity/overweight in heterozygotes for null mutations in POMC suggests that loss of one copy of POMC is sufficient to predispose to obesity (3). We and others have described a variety of heterozygous missense mutations in POMC that are found more commonly in obese children (4, 5). Arg236Gly disrupts a dibasic cleavage site between β-MSH and β-endorphin (6, 7), resulting in a β-MSH/β-endorphin fusion protein that binds to MC4R but has reduced ability to activate the receptor (6). The association of a loss of function mutation in β-MSH (Tyr221Cys) with an increased risk of obesity supports a role for this particular melanocortin peptide in the control of human energy homeostasis (4, 5).

Proopiomelanocortin (POMC) is synthesized on the rough endoplasmic reticulum and transported to the trans-Golgi network (TGN) where it is packaged into immature secretory granules of the regulated secretory pathway. Within these granules, POMC is cleaved by endoproteases to yield the active peptides, which are stored in mature (dense-core) granules to be released upon stimulation (8). There is some evidence to suggest that the N-terminal peptide of POMC carries a sorting signal motif that directs POMC to the regulated secretory pathway, although other studies have suggested this region may not be necessary for targeting to secretory granules (9, 10, 11). To date, no naturally occurring variants in this region of the human POMC gene have been described.

We now report two patients who were referred for the investigation of severe early-onset obesity and were found to be heterozygous for two different missense mutations in the N-terminal region of the POMC gene. Both mutations appear to impair the processing and sorting of POMC to the regulated secretory pathway.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Screening of the POMC gene

The POMC gene was screened for mutations in 500 subjects with severe early-onset obesity as described previously (4); all mutations were confirmed by direct nucleotide sequencing. DNA and phenotypic information was obtained from family members of the probands with N-terminal missense mutations and the genotype of relatives was ascertained by direct nucleotide sequencing. The prevalence of each mutation in over 100 alleles from an appropriate ethnically matched control group was determined by sequencing (4). Body mass index (BMI) SD scores were calculated using United Kingdom reference data (12).

Construction of POMC C28F and L37F expression vectors

Construction of a human POMC expression vector (in pcDNA3; Invitrogen Life Technologies, Carlsbad, CA) containing a c-Myc epitope tag near the carboxy terminus has been described previously (6). The epitope tag precedes the KKGE sequence to avoid removal after cleavage at the KK sequence. This construct was used as a template for mutagenesis to introduce the G to T (Cys28Phe) and C to T (Leu37Phe) mutations using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the guidelines of the supplier. Mutations were confirmed by direct nucleotide sequencing.

Biochemical properties of POMC variants

Metabolic labeling studies Mouse insulinoma β-TC3 cells were chosen for their well characterized regulated secretory pathway and lack of endogenous POMC (13). Cells (5–6 x 105) were transfected with 2 µg expression vector and 6 µl Lipofectamine 2000 (Invitrogen) and plated in 10-cm2 dishes. Cotransfections were performed using 1 µg POMC or POMC variant, together with 0.5 µg PC2 and 0.5 µg 7B2 based on previous studies (6). Metabolic labeling was performed the next day for 1 h, using 200 µCi [35S]methionine (PerkinElmer, Norwalk, CT; specific activity 1175 Ci/mmol) in 600 µl methionine-deficient RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO), and chased for 2 h in medium supplemented with excess (0.2 mM) unlabeled methionine. Cells were lysed in 1 ml lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100] containing Complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). Lysates were precleared with preformed complexes of protein G Sepharose (GE Healthcare, Piscataway, NJ) and mouse preimmune IgGs. Specific immunoprecipitation was performed with ascites produced in-house from hybridoma 9E10, directed against the c-Myc epitope. The immunoprecipitated proteins were size separated by SDS-PAGE (Criterion; Bio-Rad, Hercules, CA), visualized using the Typhoon 9400 Imager, and quantified using ImageQuant software (both GE Healthcare). The signal was divided by the number of methionines in the protein.

Studies of constitutive and regulated secretion The secretion patterns of the POMC variants were analyzed as described previously (13) with minor modifications. Briefly, cells were transfected as described above and then cultured for 16 h in serum-free medium to minimize the tonic release of POMC-derived peptides induced by serum factors. The next day, the cells were first incubated for 3 h with serum-free medium (unstimulated fraction) and subsequently for 3 h with medium containing the secretagogues forskolin (10 µM; Sigma) and isobutylmethylxanthine (IBMX) (0.1 mM; Sigma), which elevate the intracellular cAMP concentration. These relatively long incubation times were necessary to obtain detectable signals for β-endorphin in the POMC variants. After collection of this conditioned medium (stimulated fraction), the cells were lysed in Laemmli sample buffer.

Western blot analysis Culture medium from the experiment above was mixed with 25 µg/ml albumin before precipitation with 4 vol methanol at –20 C. Medium precipitates were dissolved in sample buffer and size separated by SDS-PAGE (Criterion; Bio-Rad). Western blotting was performed using monoclonal antibody 9E10 directed against the c-Myc epitope.

POMC and {alpha}-MSH immunoassay Culture media and cells from parallel experiments to those for Western blotting were collected for POMC and {alpha}-MSH analysis. Cells were lysed in 1 ml lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100] containing Complete protease inhibitors (Roche Molecular Biochemicals). {alpha}-MSH was measured by RIA (Euro-diagnostica AB, Malmö, Sweden). POMC was measured with a two-antibody ELISA (14) based on the immunoradiometric assay previously described (15). The ELISA uses two compatible monoclonal antibodies, one of which is labeled and the second of which is coated on the plates. Monoclonal antibody N1C11 recognizes the {gamma}-MSH region of POMC, whereas monoclonal antibody A1A12 recognizes the ACTH 10–18 region of POMC. The assay standards were human POMC purified from a small-cell lung cancer cell line. Assay sensitivity during the study was 10 pmol/liter.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
In addition to finding five subjects harboring previously described mutations (two with the Tyr221Cys mutation and three harboring Arg236Gly) (4, 5, 6), we identified two novel heterozygous missense mutations in exon 2 of POMC (Fig. 1AGo). C28F was found in a 17-yr-old boy of Turkish origin with severe obesity and not in 100 control alleles of the same ethnic group. This mutation was also found in two other members of the family (Fig. 1BGo), both of whom were overweight (BMI 28 and 29 kg/m2, respectively). L37F was found in a 9-yr-old girl of United Kingdom Caucasian origin with severe obesity and not in 100 control alleles of the same ethnic group. The mutation was inherited from her mother who was overweight (BMI 27 kg/m2) (Fig. 1BGo).


Figure 1
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  		FIG. 1. Screening of the POMC gene in 500 patients with severe early-onset obesity. A, Chromatograms for probands 1 and 2 indicating heterozygosity for missense mutations C28F and L37F, respectively (arrow); B, cosegregation of C28F and L37F with overweight/obesity in family members. Genotypes are included where available. N denotes normal allele, M denotes mutant allele; half-filled symbols indicate heterozygous individuals, and probands indicated with an arrow. BMI SD scores are indicated beneath genotype (12 ); BMI (kilograms per square meter) is indicated in parentheses for subjects over 16 yr of age.

 
Both residues are located in the N-terminal region of POMC (Fig. 2AGo), which has previously been implicated in the sorting of POMC to the regulated secretory pathway. Both C28 and L37 are highly conserved across multiple species (Fig. 2BGo). This region contains two disulfide bridges stabilizing an amphipathic hairpin loop structure between the second and third cysteine residues (9).


Figure 2
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  		FIG. 2. C28 and L37 are highly conserved residues in the N terminus of POMC. A, Structure of POMC and location of mutations. C28F and L37F are located in the N-terminal region of POMC distant from previously reported missense mutations (arrows) associated with obesity. B, Sequence alignment shows that Cys28 and Leu37 residues are highly conserved residues, present in the POMC sequence of species from fish to mammals.

 
To study the effect of the mutations on the maturation of POMC, biosynthetic studies were performed in transfected β-TC3 cells labeled with [35S]methionine and chased for 2 h to facilitate maturation (Fig. 3Go). Cell lysates were immunoprecipitated with an antibody directed against the carboxyl-terminal c-Myc tag and therefore recognizing the uncleaved POMC precursor and the cleavage products β-lipotropic hormone (β-LPH) and β-endorphin, the most C-terminal cleavage products. There is good evidence that β-endorphin is a marker of stimulated release from the secretory pathway (16, 17). A significant reduction in β-endorphin was observed with the C28F mutant compared with wild-type POMC. A similar but less severe reduction was also observed with the L37F mutant. These results indicate that for both POMC mutants, newly synthesized POMC is less efficiently processed into bioactive peptides than is the case with wild-type POMC.


Figure 3
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  		FIG. 3. C28F and L37F mutations impair the intracellular generation of POMC products. A, Immunoprecipitation of radiolabeled C-terminally Myc-tagged POMC-derived peptides from β-TC3 cells expressing wild-type POMC, mutant Cys28Phe POMC, or mutant Leu37Phe POMC. Processing of both POMC variants to β-endorphin is significantly reduced. ***, P < 0.001 (two-tailed t test compared with wild-type POMC). A representative image of six experiments is shown.

 
Because the amino terminus of POMC has been shown to be important for sorting of POMC into dense-core granules (10), we undertook studies designed to examine the effects of the N-terminal mutations on POMC sorting. Stimuli that promote secretion of POMC products from endocrine cells do so by promoting the fusion of dense-core granules, the most mature element of the regulated secretory pathway, with the plasma membrane, whereas secretion of precursor peptides such as POMC occurs through the stimulus-independent constitutive secretory pathway. β-TC3 cells that endogenously express PC1 and PC2 (and cotransfected with PC2 and 7B2 to improve processing efficiency as described previously) (6) were transfected with wild-type or mutant POMC and incubated in serum-free medium for 16 h to minimize tonic release of POMC by serum factors. Cells were then exposed to fresh serum-free medium for 3 h and subsequently with serum-free medium containing forskolin and IBMX for 3 h after which the conditioned medium and cell lysates was assayed for POMC and {alpha}-MSH. Both the absolute amount of {alpha}-MSH and the ratio of {alpha}-MSH/POMC were significantly reduced with the C28F mutant (Fig. 4AGo) compared with wild-type POMC. This was particularly true for the stimulated release but also markedly reduced in the medium from unstimulated cells and in the cell lysates. The results for the L37F variant displayed the same trend, but the observed differences were statistically significant only for the unstimulated medium.


Figure 4
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  		FIG. 4. Effects of C28F and L37F on constitutive and regulated secretion of POMC products. A, β-TC3 cells transfected with wild-type POMC or C28F or L37F mutations were incubated in unstimulated medium (M–) or medium with forskolin and IBMX (M+) to stimulate release of peptides from the regulated secretory pathway. Cell lysates (L) were collected after the stimulation period. {alpha}-MSH is represented as a fraction of POMC. The Cys28Phe variant resulted in much less {alpha}-MSH, and that of the Leu37Phe variant is intermediate between wild-type and Cys28Phe. n = 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed t test compared with wild-type POMC). Results are representative of four other experiments. B, Parallel transfections used the Myc-Tag on the C-terminal of POMC for Western blotting of samples from medium without secretagogues (M–) and medium with secretagogues (M+) and lysates (L). Cys28Phe POMC and to a lesser extent Leu37Phe POMC were not sorted as efficiently as wild-type POMC, with less β-LPH and β-endorphin detected. β-LPH secretion was not increased by secretagogues, indicating that it was mainly processed before sorting, and was constitutively secreted, whereas β-endorphin was mainly secreted after stimulation. Lower panel is the same experiment with longer exposure. Only 5% of cell lysates were loaded. A representative of two experiments is shown.

 
In parallel transfections, the presence of C-terminally myc-tagged POMC was used to analyze the processing by Western blotting. In the case of wild-type POMC, in the absence of a stimulus for regulated secretion, mainly POMC and some β-LPH and β-endorphin were secreted (Fig. 4BGo). After the addition of secretagogues, a large increase in the secretion of β-endorphin was observed compared with levels of secreted POMC. These results support the notion that secreted β-endorphin is an appropriate marker for POMC’s normal entry into, and processing within, the regulated secretory pathway. With the C28F mutant, the amount of POMC secreted was similar to that seen with wild type, but smaller amounts of β-LPH and β-endorphin were secreted in both conditions, consistent with the findings shown in Fig. 3AGo. Similar, but less marked, results were seen with L37F. Given the distance of the mutants from the main processing sites, the most parsimonious explanation for this is that the mutations interfere with the entry of POMC to the normal regulated secretory pathway, a finding consistent with previous studies of artificial mutants in this region of the molecule (9).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The melanocortin products of the POMC gene are involved in a wide range of biological processes from adrenal steroidogenesis to skin pigmentation (18). Products of the POMC gene expressed in a small subset of neurons in the arcuate nucleus of the hypothalamus appear to be essential for normal energy homeostasis (19) because mice and humans entirely lacking POMC develop hyperphagia and severe obesity despite lacking circulating glucocorticoids (20). Since the original description of complete human POMC deficiency (1), there have been a range of reports describing the association of heterozygous missense mutations in POMC with an increased risk of obesity (21). The vast majority of these missense mutations affect either the structure or function of {alpha}- or β-MSH or the cleavage from their precursor (22). The phenotype of obesity associated with loss-of-function mutations appears to involve an early onset of obesity and, when tested, objective evidence for increased appetite and food intake (3). Limited data were available on the carriers of the two rare mutations reported here (C28F and L37F), but both probands did have early-onset obesity, and the proband with the L37F did show increased food intake at an ad libitum test meal (data not shown) consistent with loss of melanocortin function. Although insufficient family members were available to formally assess cosegregation with these mutations, all available carriers of the mutations were either overweight or obese. Fasting plasma glucose and insulin, serum lipids, TSH, and free T4 were within the normal reference range in mutation carriers (data not shown). There were no notable abnormalities relating to the onset of puberty and no menstrual cycle irregularities in patients with impaired POMC processing, in keeping with the phenotype of POMC null and POMC haploinsufficient patients reported previously (2, 3, 4).

C28 and L37F differ notably from previous POMC mutations associated with obesity in that they do not lie within, or adjacent to, the melanocortin peptides known to have an established role in energy homeostasis (i.e. {alpha}- or β-MSH) but lie in the N-terminal region of the POMC molecule. If these mutations are causing a loss of POMC-derived signaling peptides, then they must be doing so by a novel mechanism, most likely related to sorting and processing of the POMC propeptide. Because the patients are heterozygous for these mutations, we cannot tell how far impaired POMC processing contributes to the weight phenotype given the one normal POMC allele in these patients or whether possible dominant-negative effects play a role in vivo. Because POMC sorting and processing is a topic of considerable general interest, these naturally occurring mutations may act as illuminating experiments of nature shedding some light on the sequence requirements for normal POMC targeting and processing.

The sorting of proteins to the regulated secretory pathway is still a matter of debate (23). For POMC, the 26 amino acids immediately carboxyl-terminal to the signal peptide (amino acids 27–52) were sufficient to target a reporter protein to the regulated secretory pathway in one study (10), whereas in another study, this domain was found not to be sufficient for sorting (24). Within this sequence, two disulfide bridges are formed by Cys28/Cys50 and Cys34/Cys46. The hairpin loop between Cys28 and Cys50 forms an amphipathic loop and contains highly conserved amino acids, in particular Asp36, Leu37, Glu40, and Leu44 (9) Sorting of POMC is required for efficient processing to its bioactive peptides. Its processing starts in the TGN and proceeds in the regulated secretory pathway (25). The two main enzymes involved in its processing are the proprotein convertases PC1/3 and PC2. Cleavage by PC1/3 starts in the late Golgi subcompartments/immature secretory granules and results in the generation of β-LPH and some ACTH (25). PC2 requires a more acidic environment and therefore becomes active only upon maturation of the immature secretory granules, resulting in the production of melanocortins and β-endorphin (26).

Sorting of POMC from TGN to immature secretory granules may be a key regulatory step that determines the extent of POMC processing (8). There are different theories regarding the mechanisms by which POMC is sorted and targeted to the regulated secretory pathway. Currently, the majority of studies support a sorting-for-entry model where targeting to granules is determined in the TGN (27). The amino-terminal amphipathic loop on POMC has been suggested to be an important sorting signal motif (9), possibly through binding to carboxypeptidase E as a putative sorting receptor (28). Therefore disruption of this region by changes in amino acids is likely to impede sorting to secretory granules and the effective processing of POMC to bioactive peptides. Cys28 has previously been mutated by others without observing a deleterious effect on sorting (9, 11). This discrepancy with our observations can be explained because Cys28 was replaced by the physicochemically similar amino acid serine in these studies. Here, the much larger phenylalanine is likely to cause steric hindrance and destabilize the overall structure of the hairpin loop. It is possible that the unpaired half of the disulfide bond causes endoplasmic reticulum stress, as is seen for instance in mutant forms of insulin lacking the correct complement of cysteine residues (29). However, this cannot account for the reduced processing of POMC to β-endorphin and sorting to the regulated secretory pathway, as these are post-endoplasmic reticulum events. Although mutant forms of insulin that lead to neonatal diabetes appear to be toxic to the pancreatic β-cell (30), these POMC mutations are unlikely to be similarly cytotoxic because there was no evidence, in the carriers, of the clinical syndrome of ACTH insufficiency that would inevitably result from death of the pituitary corticotrophs expressing POMC.

Leu37 is highly conserved in POMC and the replacement by Phe might have been expected to have a severe effect. The modest effect observed here might be the consequence of the experimental set-up. Sorting of POMC is a multistep process including oligomerization/aggregation in the TGN, which is independent of the N-terminal loop (31). Furthermore, the influence of each domain might be cell-type specific (32). Cell-type-specific effects on processing are for instance found in the mouse model carrying a mutation in the PC1 gene resulting in a N222D substitution (33). In this model, carboxyl-terminal processing of PC1 was much more affected in the hypothalamus than in brain and pancreas. The experiments in the current study were performed in pancreatic β-cells to circumvent the problem of endogenous POMC, but it is possible that the observed differences in sorting and {alpha}-MSH production are more pronounced in hypothalamic cells.

In summary, impairment of melanocortin signaling at secretory or receptor level is strongly associated with human obesity. C28F and L37F, although clearly rare, represent a novel mechanism of disruption of human POMC function and support the notion that the N-terminal component of human POMC is important for targeting of the propeptide to the regulated secretory pathway.


   Acknowledgments
 
We are indebted to the patients and their families for their participation and to the physicians involved in the Genetics of Obesity Study (GOOS). We thank Sandra Meulemans and Anne Warhurst for expert technical assistance.


   Footnotes
 
This study was supported by the National Institute for Health Research Cambridge Biomedical Research Centre, Medical Research Council and Diabesity (EU FP6 LSHM-CT-2003-503041) (S.O.R.), the Wellcome Trust (I.S.F. and A.W.), and Geconcerteerde onderzoeksactie van de Vlaamse gemeenschap 2008/16 (J.C.). Y.S.L. was supported by an international fellowship from the Agency for Science, Technology, and Research, Singapore.

Disclosure Statement: J.W.M.C., Y.S.L., R.L.O., M.B., A.T., D.G., J.K., S.H., A.W., S.O.R., and I.S.F. have nothing to declare.

First Published Online August 12, 2008

1 J.W.M.C., Y.S.L., and R.L.O. contributed equally to this work. Back

Abbreviations: BMI, Body mass index; IBMX, isobutylmethylxanthine; β-LPH, β-lipotropic hormone; POMC, proopiomelanocortin; TGN, trans-Golgi network.

Received May 2, 2008.

Accepted August 6, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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