Design and Characterisation of Long-R³-Insulin-Like Growth Factor-I Muteins Which Show Resistance to Pepsin Digestion

Abstract

Site-directed mutagenesis was used to construct pepsin-resistant, single-point mutations of the N-terminal extended IGF-I analogue, long-R³-IGF-I. In order to identify the most susceptible sites, the kinetics of long-R³-IGF-I digestion by purified porcine pepsin were determined. Pepsin initially cleaved the LeulO-Phell bond in the N-terminal extension peptide to generate FVN-R³-IGF-I, followed in rapid succession by cleavage at Gln15-Phe16, Tyr24-Phe25, Leu10-Val11 and Met59-Tyr60 in the IGF-I moiety. Single-point mutations at these sites were designed on the basis of the preferred cleavage bonds for pepsin, as well as amino acid substitutions less likely to disturb protein structure. These included LeulOVal, Phel6Ala, Phe25Leu, Asp53Glu and Met59Gln. All five muteins retained growth-promoting activity equivalent to or higher than that of IGF-I. In terms of pepsin susceptibility, LeulOVal and Asp53Glu were degraded as rapidly as the parent long-R³-IGF-I, Met59Gln and Phe25Leu were partially stabilised, and Phel6Ala showed a marked improvement in stability over a wide range of pepsin:substrate ratios. Accordingly, the Phel6Ala mutein, long-R³A¹⁶-IGF-I, has potential for oral applications to enhance gastric growth and repair.

Key Words:

• site-directed mutagenesis
• IGF-I muteins
• pepsin resistance
• kinetics of degradation
• receptor binding
• biological activity