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Pegylated Human Leptin D23L Mutant - Preparation and Biological Activity In Vitro and in Vivo in Male ob/ob Mice.
Endocrinology 2019 Februrary 26
Recombinant monomeric human leptin (hLEP) and its D23L mutant were prepared in Escherichia coli and pegylated at their N-terminus using methoxy PEG-propionaldehyde 20 kDa. As determined by both SDS-PAGE and size-exclusion chromatography, the pegylated proteins consisted of >90% mono-pegylated and less than 10% of double-pegylated species. Circular dichroism spectra showed that their secondary structure, characteristic of all four α-helix bundle cytokines, was not affected by either the D23L mutation or pegylation. Due to the D23L mutation, affinity for hLEP receptor increased 25- and 40-fold for the pegylated and non-pegylated mutant, respectively. However, whereas the proliferation-promoting activity in vitro of both non-mutated and mutated non-pegylated hLEP was identical, that of the respective pegylated mutant was ∼6-fold higher compared to the pegylated non-mutated hLEPn. This difference was also seen in vivo. Both pegylated hLEPs at all doses significantly decreased body weight and food consumption as compared to the vehicle-treatment control. Once-daily administration of pegylated hLEP D23L at doses of 0.1, 0.3 and 1 mg/kg for 14 consecutive days in ob/ob mice significantly decreased body weight and food consumption as compared to respective pegylated hLEP-treated animals, with the biggest difference observed at 0.1 mg/kg. Repeated administration of either pegylated hLEP D23L or pegylated hLEP significantly decreased blood glucose levels compared to the control both prior to glucose challenge and following oral glucose tolerance test, but with no difference between the two treatments. In conclusion, the pegylated hLEP D23L mutant seems to be a novel, more potent reagent suitable for in-vivo studies as compared to the pegylated non-mutated hLEP.
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