Structure-function studies of human leptin

To elucidate the structural requirement of human leptin for its functions, the mild-type, mutant type, C-terminal deletion, and N-terminal deletion we re expressed in Escherichia coil and purified in soluble forms. These lepti n analogs were intracerebroventrically injected into C57BL/6J ob/ob mice, a nd their in vivo biological activities were evaluated. The mutant-type lept in lacking a C-terminal disulfide bond reduced food intake at doses of more than 15 pmol/mouse, which was as effective as the wild-type leptin, C-term inal deletion without the loop structure, also significantly, but to a less er extent, reduced food intake at doses of more than 90 pmol/mouse. However , N-terminal deletions showed no effect on food intake. We also evaluated t he effects of the leptin analogs on radiolabeled leptin binding to its rece ptor in the choroid plexus using autoradiography. An excess of unlabeled mu tant-type leptin as well as wildtype leptin led to complete inhibition of b inding. C-terminal deletions led to weak inhibitory activity, whereas N-ter minal deletions caused no inhibitory activity. These results clearly demonstrate that the N-terminal region of leptin is e ssential for both its biological and receptor binding activities. The amino acid sequence of the C-terminal loop structure is also important for enhan cing these actions, whereas the C-terminal disulfide bond is not needed.