Characterization of genomic sequence coding for bromelain inhibitors in pineapple and expression of its recombinant isoform.
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.
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.
Muramatsu
Tomonari
T
Hatano
Ken-ichi
K
Nagata
Koji
K
Tanokura
Masaru
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001963:Bromelains / Q000037:antagonists & inhibitors*; D002942:Circular Dichroism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
C095959:BI-VI protein, Ananas comosus; D015853:Cysteine Proteinase Inhibitors; D010446:Peptide Fragments; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D001963:Bromelains