Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases

Abstract

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast a-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein.Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 3 1, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 3 1. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Factor X Fusion Proteins: Improved Production and Use in the Release in Vitro of Biologically Active Hirudin from an Inactive α-Factor–Hirudin Fusion Protein

“…This sequence is consistent with the intracellular processing of residues 1-64 of the ␣-factor leader peptide, resulting from the cleavage of the bond between Leu64 and Phe65. Interestingly, the N-terminal sequence found in the secreted precursor was the same as the one reported for a secreted ␣-factor-hirustatin fusion containing a wild-type ␣-factor leader sequence (29). Although the N-terminal extension does not affect the activity of hirustatin (29), its presence appears to abolish the inhibitory activity of the secreted ␣-factor-hirudin fusion protein.…”

Production and Characterization of Recombinant Guamerin, an Elastase-Specific Inhibitor, in the Methylotrophic Yeast Pichia pastoris

“…Primary structural and X-ray crystallographic studies of antistasin and hirustasin have suggested that antistasin-type inhibitors have a similar disulfide bond pattern or tertiary structure (4,32,33). However, guamerin has a unique inhibition property toward elastase that is distinct from other antistasin-type inhibitors.…”

Amino acid sequence of piguamerin, an antistasin‐type protease inhibitor from the blood sucking leech Hirudo nipponia

“…The importance of the reactive-site loop was partially demonstrated by the inhibitory activity of a disulRecently, X-ray crystallography of the hirustasin · tissue kallikrein complex [18,19] and the antistasin · factor Xa complex fide-bridge formation consisting of 19-residue peptides that contained the reactive-site sequence of guamerin [22]. Piguamerin [20,21] revealed the molecular interactions of the inhibitors with target proteases.…”