Characterization of Genomic Sequence Coding for Bromelain Inhibitors in Pineapple and Expression of Its Recombinant Isoform

Yoriko Sawano,Tomonari Muramatsu,Ken-Ichi Hatano,Koji Nagata,Masaru Tanokura

doi:10.1074/jbc.m202600200

Yoriko Sawano, Tomonari Muramatsu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m202600200

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Abstract

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem (Reddy, M. N., Keim, P. S., Heinrikson, R. L., and Kézdy, F. J. (1975) J. Biol. Chem. 250, 1741-1750). It consists of eight isoinhibitors, and each isoinhibitor has a two-chain structure. In this study, the genomic DNA has been cloned and found to encode a precursor protein with 246 amino acids (M(r) = approximately 27,500) containing three isoinhibitor domains (BI-III, -VI, and -VII) that are 93% identical to one another in amino acid sequences. The gene structure indicated that these isoinhibitors are produced by removal of the N-terminal pre-peptide (19 residues), 3 interchain peptides (each 5 residues), 2 interdomain peptides (each 19 residues), and the C-terminal pro-peptide (18 residues). Moreover, all the amino acid sequences of bromelain isoinhibitors could be explained by removal of one or two amino acids from BI-III, -VI, and -VII with exopeptidases. A recombinant single-chain BI-VI with and without the interchain peptide showed the same and no bromelain inhibitory activity as compared with the native BI-VI, respectively. These results indicate that the interchain peptide plays an important role of the folding process of the mature isoinhibitors.

Highlights

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem
The structural similarity of BI-VIN to Birk serine proteinase inhibitor (BBI) indicates that BI and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution
We isolated the structural gene of the BI precursor protein (BIP) from pineapple leaf

Summary

Introduction

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem A recombinant single-chain BI-VI with and without the interchain peptide showed the same and no bromelain inhibitory activity as compared with the native BI-VI, respectively These results indicate that the interchain peptide plays an important role of the folding process of the mature isoinhibitors. Most cysteine proteinase inhibitors obtained from plants so far have been identified as cystatins including inhibitors from rice, corn, soybean, and tomato [1] Their properties have been extensively studied, including their nucleotide and amino acid sequences and three-dimensional structures. We isolated the genomic DNA encoding a precursor protein of BI (BIP) from pineapple leaf and sequenced it to elucidate the molecular evolution and functional expression of the two-chain inhibitor BI. We proposed a model mechanism of the putative processing from BIP to the mature isoinhibitors (Fig. 8)

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