Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists.
Expression plasmids encoding mouse and rat leptins and their L39A/D40A/F41A muteins were prepared. The proteins were expressed in Escherichia coli, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric protein. Circular dichroism (CD) analysis revealed that the mutations hardly affect the leptins' secondary structure, and they were similar to previously reported CD spectra for human leptin. Both mouse and rat leptins were biologically active in promoting proliferation in BAF/3 cells stably transfected with the long form of human leptin receptor. The mutations did not change the binding properties to BAF/3 cells as compared, respectively, to non-mutated mouse, rat or human leptins, or their ability to form 1:1 complexes with the leptin-binding domain of chicken leptin receptor. In contrast, their biological activity, tested in a BAF/3 proliferation assay, was abolished and both became potent antagonists. As the LDF (amino acids 39-41) sequence is preserved in all known leptins, the present results substantiate the hypothesis that this sequence plays a pivotal role in leptins' site III and that interaction of leptin with its receptors resembles the corresponding interactions of interleukin-6 and granulocyte colony-stimulating factor their receptors.
Salomon
Gili
G
Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, Rehovot 76100, Israel.
Niv-Spector
Leonora
L
Gussakovsky
Eugene E
EE
Gertler
Arieh
A
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D020738:Leptin / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000235:genetics*; D007930:Leucine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D020033:Protein Isoforms / Q000819:agonists / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000235:genetics
D020738:Leptin; D020033:Protein Isoforms; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010649:Phenylalanine; D007930:Leucine; D000409:Alanine