ANALYSIS OF SERPIN INHIBITORY FUNCTION BY MUTAGENESIS OF OVALBUMIN AND GENERATION OF CHIMERIC OVALBUMIN PAI-2 FUSION PROTEINS

Ovalbumin is a non-inhibitory serpin which lacks the ability to underg o the S --> R transition or conformational change. Amino acid residues in the hinge region (P-11 to P-14) of ovalbumin and other non-inhibit ory serpins differ from the concensus sequence of this region of inhib itory serpins, and have been proposed to be responsible for lack of in hibitory properties, particularly the P-14 charged residue. Site direc ted mutagenesis using PCR overlap extension was performed on these res idues in ovalbumin to create a mutant with three amino acid changes, R 340T, V342A and V343A. However analysis of the mutant recombinant oval bumin with the consensus residues failed to show inhibitory activity o r decreased stability, indicating that the hinge region alone is not r esponsible for lack of inhibition. A series of three fusion proteins w ere then constructed by replacing varying C-terminal regions of ovalbu min with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. Thi s resulted in the replacing of structural features such as the reactiv e site loop, hinge region and beta sheet strands 5A and 6A. However bo th fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition o f uPA, SDS-PAGE stable complex formation with uPA and increased instab ility on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may h ave a key role in serpin beta-sheet opening and inhibitory function. ( C) 1997 Academic Press Limited.