Susceptibility of the interchain peptide of a bromelain inhibitor precursor to the target proteases bromelain, chymotrypsin, and trypsin.
Bromein, a cysteine proteinase inhibitor from pineapple stem, is a unique double-chain inhibitor. The 27.5-kDa precursor protein is processed by the removal of three interchain, two interdomain, and two terminal-flanking peptides, thus resulting in the release of mature isoinhibitors of approximately 6 kDa. To characterize the processing of the interchain peptide Thr15-Ser-Ser-Ser-Asp, we expressed a single-chain precursor with this peptide and monitored proteolytic cleavage by the target proteinase bromelain. By peptide sequencing and mass spectrometric analysis, the initial cleavage was found to occur in vitro between the light-chain and interchain peptides; subsequent trimming formed the terminal-ragged peptides Thr15-Lys60, Ser17-Lys60, Ser18-Lys60, and Asp19-Lys60. However, bromelain did not show any cleavage activity between the interchain and heavy-chain peptides. We also discovered that cleavage between the light-chain and interchain peptides is essential for the single-chain inhibitor to exhibit full inhibitory activity. Notably, the incompletely processed intermediates showed higher inhibitory activity than either the native bromein or the single-chain precursor. Bromein is also known to weakly inhibit the serine proteinases chymotrypsin and trypsin; however, a recombinant single-chain inhibitor with the interchain peptide was no longer able to inhibit these serine proteinases.
Sawano
Yoriko
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Hatano
Ken-ichi
K
Tanokura
Masaru
M
D001963:Bromelains / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry*; D006868:Hydrolysis; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011498:Protein Precursors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry*
C095959:BI-VI protein, Ananas comosus; D010455:Peptides; D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D001963:Bromelains; D002918:Chymotrypsin; D014357:Trypsin