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 undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin 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. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both 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 of uPA, SDS-PAGE stable complex formation with uPA and increased instability 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 have a key role in serpin beta-sheet opening and inhibitory function.
McCarthy
B J
BJ
Department of Biochemistry, University College Dublin Belfied, Ireland.
Worrall
D M
DM
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010047:Ovalbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017396:Plasminogen Activator Inhibitor 2; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D010047:Ovalbumin