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.
Highlights
Of new therapeutics with restricted inhibitory activities that selectively target individual proteases has the potential to both improve the efficacy and alleviate toxicities in proteasetargeting treatment strategies
We have produced and characterized 12 variant A2Ms in order to elucidate the effects of the tabula rasa bait region substitution and demonstrate an approach to developing new protease inhibitors, which selectively inhibit a target protease
Wild-type A2M is an efficient protease inhibitor with an extensive inhibitory repertoire, and it is well established that A2M can inhibit new proteases when an appropriate substrate sequence is added into the bait region [26,27,28]
Summary
Of new therapeutics with restricted inhibitory activities that selectively target individual proteases has the potential to both improve the efficacy and alleviate toxicities in proteasetargeting treatment strategies. This tabula rasa bait region was not initially cleaved by any of 12 tested proteases, but could be cleaved by trypsin, LysC, or MMP2 upon introducing an appropriate cleavage site into its sequence.
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