Functional characterization of a cystatin A from the bat Myotis davidii

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the...

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Published in:Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2024-10, Vol.274, p.111003, Article 111003
Main Authors: Costa, Gabriel Cerqueira Alves, Torquato, Ricardo Jose Soares, de Morais Gomes, Vinícius, Rosa-Fernandes, Lívia, Palmisano, Giuseppe, Tanaka, Aparecida Sadae
Format: Article
Language:English
Subjects:
Cathepsins
Cystatins
Myotis davidii
Stefins
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Summary:Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73–79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases. [Display omitted] •Myotis davidii's cystatin A is a strong inhibitor of papain, cathepsins L and B.•Cystatin A from Myotis davidii and Homo sapiens differ at the N-terminal and second binding loop.•Myotis davidii's cystatin A has a 20 times lower Ki for cathepsin B than human cystatin A.
ISSN:1096-4959
1879-1107
1879-1107
DOI:10.1016/j.cbpb.2024.111003