Rapid Identification of Highly Active and Selective Substrates for Stromelysin and Matrilysin Using Bacteriophage Peptide Display Libraries

Abstract

The discovery of useful peptide substrates for proteases that recognize many amino acids in their active sites is often a slow process due to the lack of initial substrate data and the expense of analyzing large numbers of peptide substrates. To overcome these obstacles, we have made use of bacteriophage peptide display libraries. We prepared a random hexamer library in the fd-derived vector fAFF-1 and included a "tether" sequence that could be recognized by monoclonal antibodies. We chose the matrix metalloproteinases stromelysin and matrilysin as the targets for our studies, as they are known to require at least 6 amino acids in a peptide substrate for cleavage. The phage library was treated in solution with protease and cleaved phage separated from uncleaved phage using a mixture of tether-binding monoclonal antibodies and Protein A-bearing cells followed by precipitation. Clones were screened by the use of a rapid screening assay that identified phage encoding peptide sequences susceptible to cleavage by the enzymes. The nucleotide sequence of the random hexamer region of 43 such clones was determined for stromelysin and 23 for matrilysin. Synthetic peptides were prepared whose sequences were based on some of the positive clones, as well as consensus sequences built from the positive clones. Many of the peptides have kcat/KM values as good or better than those of previously reported substrates, and in fact, we were able to produce stromelysin and matrilysin substrates that are both the most active and smallest reported to date. In addition, the phage data predicted selectivity in the P2 and P'1 positions of the two enzymes that were supported by the kinetic analysis of the peptides. This work demonstrates that the phage selection techniques enable the rapid identification of highly active and selective protease substrates without making any a priori assumptions about the specificity or the "physiological substrate" of the protease under study.

Substrate Hydrolysis by Matrix Metalloproteinase-9*

“…Significant differences are also observed at P 2Ј . In the group I MMP-9 substrates, 23 of 28 substrates have Ser or Thr at P 2Ј , but neither residue is prevalent at this subsite in the substrates selected for the other MMPs (32,47). These observations support the contention that subsite interactions other than P 3 and P 1Ј have a significant impact on substrate selectivity among the MMP family.…”

“…Although Arg can also be found at P 2 in peptide substrates for MMP-13, its frequency is much lower than in the MMP-9 substrates we selected (47). Furthermore, Arg is rarely, if ever, found at P 2 in MMP-3 or -7 peptide substrates (32). Ser or Thr most frequently occupies the P 1 position of the MMP-9 substrates.…”

“…The phage-antibody complexes were precipitated by the addition of 100 l of Pansorbin (Calbiochem). The cleaved phage remaining in the supernatant were amplified using K91 E. coli as described previously (32)(33)(34) and were then used for subsequent rounds of substrate selection.…”

Gelatinase-mediated migration and invasion of cancer cells

“…These phage can then be amplified and further enriched to determine the optimal substrates for the protease of interest. This technique has been used to map the cleavage sites of subtilisin (Matthews and Wells, 1993), furin (Matthews et al, 1994) and several matrix metalloproteinases (MMPs) Deng et al, 2000;Kridel et al, 2001;Kridel et al, 2002;Smith et al, 1995). Not only protease substrates can be found using phage display.…”

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

“…Although the protein (pIII) encoded by gIII is chymotrypsin-, thermolysin-, and subtilisin-susceptible, it is trypsin-resistant (31). Thus, phage display peptide libraries can give insight into the substrate specificities of certain proteases (32,33). The N terminus of pIII extends out from the surface of the body of the filamentous phage.…”