Engineering and investigation of protease fine specificity

Date
2010-12
Authors
Li, Haixin
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Abstract

The Escherichia coli (E. coli) outer membrane protease OmpT is an endopeptidase of the omptin family in gram negative bacteria. OmpT cleave preferentially between two consecutive basic residues, especially Arg-Arg, and it has been classified as an aspartyl protease based on its crystal structure although biochemical confirmation of a catalytic aspartyl residue is lacking (Vandeputte-Rutten, et al., 2001). Our lab has successfully engineered the P1 and P1’ specificity and selectivity of OmpT by employing novel strategies for the isolation of enzyme variants that cleave desired substrates from large combinatorial libraries screened by flow cytometry. However, the engineering of proteases with altered specificity beyond the P1 and P1’ residues of the substrate have not been demonstrated. By applying high throughput screening of large libraries of OmpT constructed by structure-guided saturation mutagenesis of the S2 subsite (which recognizes the P2 residue), as well as random mutagenesis by error prone viii PCR and DNA shuffling, we engineered an OmpT variant exhibiting about 56 fold change in the selectivity for the P2 position in peptide substrates. Specifically, this enzyme preferred an acidic residue (Glu) over Tyr which is preferred by the wild type OmpT. Molecular modeling was then employed to provide insights on how mutations in OmpT mediated this change in P2 specificity. A long term goal of protease engineering is to generate highly specific enzyme variants that can be used for the irreversible inactivation of disease targets. The anaphylatoxin C3a is a key mediator in inflammation and has been implicated with multiple inflammatory diseases. Since the site of anaphylatoxin C3a recognized by cellular receptors lie in its C-terminus, a protease cleaving the C-terminus of C3a could be therapeutically relevant. Using high throughput screening and directed evolution we successful isolated C3a cleaving enzyme variants and have characterized them biochemically. Finally as part of this dissertation we have employed high throughput screening methods to dissect the substrate specificity of members of the kallikrein family of mammalian proteases which are implicated in a number of physiological and disease functions. The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction and vascular cell growth. KLK6 is over-expressed in breast and ovarian cancer tissues and has been shown to cleave peptides derived from human ix myelin protein and the Aβamyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6 by substrate phage-display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at the P1 site, Ser/Arg strongly preferred at P1’ and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1’. Docking simulations of consensus peptide substrates was used to infer possible identities of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates such as ionotropic glutamate receptor (GluR) and synphilin.

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