The tissue-type plasminogen activator inhibitor ETIa from Erythrina variegata: Structural basis for the inhibitory activity by cloning, expression, and mutagenesis of the cDNA encoding ETIa

Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA). A cDNA clone encoding ETIa was isolated from the λgt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing. The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids. Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively. The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant ETIa (rETIa) was expressed in E. coli as inclusion bodies; it was purified to homogeneity by gel filtration on Sephadex G-75. The rETIa exhibited almost the same inhibitory activity toward trypsin and tPA as ETIa. Six mutants, in which the amino acids Arg⁶¹, Leu⁶²) Arg⁶³, and Ala⁶⁵ were replaced by Pro, Phe, Leu/Asp, and Tyr, respectively, were constructed by site-specific mutagenesis and expressed in E. coli. The site-specific mutation of Arg⁶³ to Leu (aR63L) or Asp (aR63D) in ETIa resulted in abolition of the inhibitory activities toward both trypsin and tPA. The mutants aR61P and aL62F showed significantly reduced tPa-inhibitory activity, and furthermore the double mutant aR61P/L62F lacked tPA-inhibitory activity, despite retaining the trypsin-inhibitory activity. In contrast, the mutant aA65Y exhibited tPA-inhibitory activity to the same extent as rETIa. This result suggests that Arg⁶¹ and Leu⁶² in ETIa, in addition to Arg⁶³, may play an important role in the interaction with tPA.