Bioengineering of Human Thyrotropin Superactive Analogs by Site-directed “Lysine-scanning” Mutagenesis

This is the authors' abstract. We don't add key points for this paper.

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the αL3 loop of the common α-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent αL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the αL3 loop analog compared with both the αL1 loop analog and the hTSH-βL3 loop analog was observed. We demonstrated that spatially distinct regions of the common α-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.