Truncated human leptin (Δ133) associated with extreme obesity undergoes proteasomal degradation after defective intracellular transport

We recently described a homozygous frameshift mutation in the human leptin (ob) gene associated with undetectable serum leptin and extreme obesity in two individuals (1). This represented the first identified genetic cause of morbid obesity in humans. Preliminary data suggested a defect in the secretion of this truncated (Delta 133) mutant leptin. In the present investigation, we have examined the mechanisms underlying the defective secretion of the Delta 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 Delta 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 p-lactone, led to a significant increase in the intracellular levels of Delta 133 leptin, indicating a role for the proteasome in the degradation pathway. Although intracellular immunoprecipitated wt and Delta 133 leptin levels were comparable, analysis of total cell lysates revealed a 7-fold increase in total intracellular Delta 133 leptin, compared with wt leptin. Size-exclusion membrane filtration demonstrated that intracellular Delta 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 Delta 133 leptin was detected by immunofluorescence microscopy. In conclusion, the Delta 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.