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Determination of protease cleavage site motifs using mixture-based oriented peptide libraries

Nature Biotechnology volume 19, pages 661–667 (2001)Cite this article

Abstract

The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates.

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Figure 1
Figure 2: Cleavage-site specificity of MMP-7 (matrilysin).
Figure 3: MMP-2 can act as a neurocan-processing enzyme in vitro.

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Acknowledgements

Purified neurocan was a generous gift from Richard U. Margolis of the Department of Pharmacology, New York University Medical Center. The 1F6 anti-neurocan monoclonal antibody developed by Renee K. Margolis and Richard U. Margolis was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa Department of Biological Sciences. We thank Michael Yaffe, Thomas McGarry, Reuben Shaw, and Seth Field for helpful comments on the manuscript. We acknowledge Michael Yaffe and John V. Frangioni for initiating work in the lab on proteases with peptide libraries and for helpful discussions. This work was supported by grants from the National Institutes of Health (GM56203, and NRSA fellowship GM19895-01 to B.E.T.)

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  1. Department of Medicine, Department of Cell Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, 02215, MA

    Benjamin E. Turk, Lisa L. Huang, Elizabeth T. Piro & Lewis C. Cantley

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Correspondence to Lewis C. Cantley.

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Turk, B., Huang, L., Piro, E. et al. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat Biotechnol 19, 661–667 (2001). https://doi.org/10.1038/90273

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