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Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes
Published on: October 3, 2019
Substrate specificity of human kallikreins 1 and 6 determined by phage display
Hai-Xin Li1, Bum-Yeol Hwang, Gurunathan Laxmikanthan
1Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Insights
This study reveals the substrate specificities of human tissue kallikrein 1 (KLK1) and KLK6 using phage display. KLK1 shows broad selectivity, while KLK6 prefers arginine at P1, offering insights into their distinct roles.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The human tissue kallikrein (KLK) family comprises 15 serine proteases with diverse physiological roles.
- KLK1 regulates blood pressure and vascular functions.
- KLK6 is implicated in breast and ovarian cancers and cleaves myelin and amyloid peptides.
Purpose of the Study:
- To determine the substrate specificity of KLK1 and KLK6.
- To identify key residues involved in substrate binding.
- To predict novel protein substrates for KLK6.
Main Methods:
- Substrate phage display with a random octapeptide library.
- Analysis of cleavage preferences at specific amino acid positions (P1, P1', P2).
- Molecular docking simulations and bioinformatic analysis.
Main Results:
- KLK1 exhibits trypsin- and chymotrypsin-like activity, preferring Tyr/Arg at P1 and Ser/Arg at P1'.
- KLK6 demonstrates trypsin-like activity, exclusively cleaving after Arg at P1 and preferring Ser at P1'.
- Docking simulations identified enzyme residues crucial for substrate recognition.
Conclusions:
- Distinct substrate specificities of KLK1 and KLK6 elucidated.
- Identified potential KLK6 substrates include ionotropic glutamate receptors and synphilin.
- Provides a foundation for understanding KLK functions in health and disease.
Abstract:
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 overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Abeta amyloid 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 site P1, 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 provide information on the identity 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.
