Substrate specificity of human kallikreins 1 and 6 determined by phage display

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 Ab 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 P19, 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 P19. 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.

Novel Biological Substrates of Human Kallikrein 7 Identified through Degradomics

“…In addition to yeast two-hybrid, chemical or biological peptide library scanning has also been used to investigate substrate cleavage sites for several kallikreins. For example, substrates for KLK1, KLK2, KLK3-7, KLK10 -11, and KLK14 have been determined with the use of chemical peptide library scanning (29 -32), whereas specificity and potential biological targets for KLK1, KLK2, KLK4, KLK6, and KLK14 have been scanned by phage display screening (33)(34)(35). Some of the limitations of these approaches include detection of interaction but not activity per se, use of short peptides (not whole proteins), and detection of substrates under non-native conditions.…”

Unleashing the therapeutic potential of human kallikrein-related serine proteases

“…Several techniques have been applied to characterize the preferred prime-side and non-prime-side substrate specificities of individual KLKs, including phage display (which has been used to characterize KLK1-KLK3, KLK6 and KLK14) [25][26][27][28][29] , positional scanning of synthetic combinatorial libraries (PS-SCL; used for KLK3-KLK7, KLK10, KLK11, KLK13 and KLK14) [30][31][32][33] and peptide screening (used for KLK1-KLK3, KLK6, KLK8 and KLK12-KLK14) [34][35][36][37][38][39][40][41] . As shown in Supplementary information S2 (table), trypsin-like KLKs (that is, KLK2, KLK4-KLK6, KLK8 and KLK11-KLK14) predominantly cleave the peptide bond when the carboxyl side of the amide bond at the P1 position of the substrate is a positively charged residue, such as an arginine or lysine.…”

5 Molecular Recognition Properties of Kallikrein-related Peptidases on Synthetic and Endogenous Substrates

“…KLK1 displays trypsin-and chymotrypsin-like specificity with both endogenous and synthetic substrates. The processing of kininogen-1 to kallidin requires the cleavage of the Met 379 ↓Lys 380 and Arg 389 ↓Ser 390 bonds by KLK1 (Li et al, 2008). To understand the requirements behind the hydrolysis of kininogen-1 by KLK1, Del Nery et al synthesized a number of internally quenched fluoro- From: 1, ; 2, (Borgoño et al, 2007a); 3, (Matsumura et al, 2005) Brought to you by | Uni of South Australia Library Authenticated Download Date | 10/12/15 12:37 PM genic substrates, based on the kininogen-1 residues 370-399 (Del Nery et al, 1995;Pimenta et al, 1999).…”

“…Cleavage analysis of the Arg 389 ↓Ser 390 bond in fluorogenic substrates documented important amino acid preferences in the P1′-P3′ positions that resulted in trypsin-like activity. Recently, the substrate specificity of KLK1 was determined from octapeptide sequences isolated using phage display (Li et al, 2008). From sixteen isolated clones, twelve phage clones were found to encode chymotrypsin-like substrate sequences with large hydrophobic amino acids in the P1 position.…”

“…In the P3 and P4 positions, however, KLK6 was not selective. More recently, an octapeptide phage display library has been employed to elucidate the substrate specificity of KLK6 (Li et al, 2008). All of the cleavage sequences had a P1 Arg and most had either a Phe or Val residue in the P2.…”