Development of selective protease inhibitors <i>via</i> engineering of the bait region of human α₂-macroglobulin

Harwood, Seandean Lykke, Nielsen, Nadia Sukusu, Diep, Khang, Jensen, Kathrine Tejlgard, Nielsen, Peter Kresten, Yamamoto, Kazuhiro ORCID: 0000-0002-8481-775X and Enghild, Jan J (2021) Development of selective protease inhibitors <i>via</i> engineering of the bait region of human α₂-macroglobulin. JOURNAL OF BIOLOGICAL CHEMISTRY, 297 (1). 100879-.

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Abstract

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.

Item Type:	Article
Uncontrolled Keywords:	Humans, Trypsin, Pregnancy-Associated alpha 2-Macroglobulins, Recombinant Proteins, Protease Inhibitors, Protein Engineering, Binding Sites, Substrate Specificity, Matrix Metalloproteinase 2, HEK293 Cells
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences
Depositing User:	Symplectic Admin
Date Deposited:	02 Jul 2021 10:27
Last Modified:	18 Oct 2023 11:38
DOI:	10.1016/j.jbc.2021.100879
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3128536