Structures of the free and inhibitors-bound forms of bromelain and ananain from Ananas comosus stem and in vitro study of their cytotoxicity

Mohamed Azarkan,Rafaèle Calvo Esposito,Raphael Herman,Erik Maquoi,Nasiha M'Rabet,François Delbrassine,Paulette Charlier,Frédéric Kerff

doi:10.1038/s41598-020-76172-5

Mohamed Azarkan, Rafaèle Calvo Esposito + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-76172-5

Copy DOI

Abstract

The Ananascomosus stem extract is a complex mixture containing various cysteine proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 μM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.

Highlights

The Ananas comosus stem extract is a complex mixture containing various cysteine proteases of the C1A subfamily, such as bromelain and ananain
One of the key challenges faced by researchers studying cysteine proteases, those of plant origin, was the characterization of multiple enzymeforms, such as those found in A. comosus stem e xtract[8,9]
We have recently purified to homogeneity and characterized several catalytically competent species from A. comosus stem extracts by using an efficient strategy based on the covalent grafting of an activated polyethylene glycol chain followed by purification on classical chromatographic gel media

Summary

Introduction

The Ananas comosus stem extract is a complex mixture containing various cysteine proteases of the C1A subfamily, such as bromelain and ananain. Orlandi-Mattos et al highlighted the importance of performing further biochemical and pharmacological studies by using pure and well characterized components to clearly understand the biological effects of the commercially available A. comosus stem extract sold as stem b romelain[20] These authors showed that while low concentration of A. comosus stem extract mimics the prohormones convertases 1 and 2 cleavage pattern of proenkephalin, high bromelain concentration induces additional cleavage at non-specific sites. They attributed such difference in the cleavage pattern to the minor proteolytic constituent ananain, a cysteine protease with broad specificity. This paradoxical effect should be linked to the observation that while A. comosus stem extract administered orally showed effectiveness in reducing ecchymosis[31,32], the assessment of the dose of this complex mixture to be used remains a critical factor

Methods

Results

Conclusion