title: Directed evolution towards protease-resistant hirudin variants.
abstract: Hirudin, a thrombin-specific inhibitor, is efficiently digested and inactivated by proteases with pepsin- and chymotrypsin-like specificity. Using a combination of phage display selection and high-throughput screening methods, several variants of recombinant hirudin were generated. Only very few variants comprising amino acid substitutions in the amino-terminal domain (residues 1-5) and in the carboxyl-terminal tail (residues 49, 50, and/or 56, 57, 62-64) were identified that showed thrombin inhibition activities similar to those of the wild-type polypeptide. Analysis of protease susceptibility, however, revealed that mutations, which conferred protease resistance, simultaneously diminish thrombin inhibition activity. This is particularly apparent for substitutions in the region of residues 56-64, which forms a large number of electrostatic and hydrophobic interactions with thrombin in the crystal structure of the complex. Unlike wild-type hirudin, the variant comprising Pro(50)- ...-His(56)-Asp(57)- ...-Pro(62)-Pro(63)-His(64) is completely resistant to pepsin and chymotrypsin cleavage; however, this is at the expense of thrombin inhibition activity where there is a 100-fold increase in the IC50 value. The frequent replacement of wild-type amino acids by proline at major protease cleavage sites indicates that at least pepsin- and chymotrypsin-like enzymes may exhibit a (conformational) specificity concerning the P1 and P2 positions. On the basis of these results, proline substitutions appear to be a general strategy to design polypeptides that are not susceptible to digestion by a broader range of different proteases.
authors.affiliation: Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstr. 8, 37077, Goettingen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Wirsching
authors.forename: Martina
authors.initials: M
authors.lastname: Keller
authors.forename: Christian
authors.initials: C
authors.lastname: Hildmann
authors.forename: Daniel
authors.initials: D
authors.lastname: Riester
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwienhorst
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism*; D019143:Evolution, Molecular; D015723:Gene Library; D005821:Genetic Techniques; D006629:Hirudins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D009154:Mutation; D010434:Pepsin A / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D006629:Hirudins; D011480:Protease Inhibitors; D010450:Endopeptidases; D013917:Thrombin; D010434:Pepsin A