Co- and post-translational processing of the hevein preproprotein of latex of the rubber tree (Hevea brasiliensis)
Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.
Lee
H I
HI
Department of Energy, Michigan State University, East Lansing 48824-1312.
Broekaert
W F
WF
Raikhel
N V
NV
Lee
H
H
D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D015153:Blotting, Western; D002686:Chitin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014197:Trees
D023181:Antimicrobial Cationic Peptides; D037121:Plant Lectins; D010940:Plant Proteins; D011498:Protein Precursors; C045875:hevein; D002686:Chitin