New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family.

Irina Y Filippova,Nikita I Zhiganov,Nikolay I Sokolenko,Yakov E Dunaevsky,Elena A Dvoryakova,Brenda Oppert,Elena N Elpidina,Tatiana R Simonyan,Valeriia F Tereshchenkova,Mikhail A Belozersky

doi:10.3389/fmolb.2020.578758

Abstract

New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA (p-nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family.

Highlights

Cysteine peptidases constitute one of the main clans of proteolytic enzymes and are characterized by the presence of a Cys residue in the active center
The structure of the newly synthesized substrates can be expressed by the general formula Glp-Phe-Gln↓X where Glp is pyroglutamyl; X = pNA (p-nitroanilide) or AMC (4-amino7-methylcoumaride); the location of the hydrolysis site is indicated by the arrow
The structure of the substrates meets the requirements of the specificity of cysteine peptidases from the C1 family: a key P1 position occupies a medium sized Gln residue, and in position P2, which is decisive for the specificity of these peptidases, is a hydrophobic Phe residue

Summary

Introduction

Cysteine peptidases constitute one of the main clans of proteolytic enzymes and are characterized by the presence of a Cys residue in the active center. Based on the MEROPS classification, the most numerous cysteine peptidase family is the papain C1 family (Rawlings et al, 2018). In the post-genomic era, C1 peptidases have been found in a variety of organisms – from viruses and protozoa to humans. The ancestor of the family is the plant peptidase papain, most attention has been paid to mammalian and human cysteine peptidases, called cathepsins. 11 human cysteine cathepsins are known: cathepsins B, C, F, H, K, L, O, S, L2 (alternative name V), X, and W, and the genes encoding these enzymes were found in a bioinformatics analysis of the human genome (Rossi et al, 2004). The main function of cysteine cathepsins is the non-specific

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