Posttranslational modification of an isoinhibitor from the potato proteinase inhibitor II gene family in transgenic tobacco yields a peptide with homology to potato chymotrypsin inhibitor I.
Abstract
A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.
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- Atkinson AH, Heath RL, Simpson RJ, Clarke AE, Anderson MA. Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell. 1993 Feb;5(2):203–213.[PMC free article] [PubMed] [Google Scholar]
- Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 1984 Nov 26;12(22):8711–8721.[PMC free article] [PubMed] [Google Scholar]
- Bryant J, Green TR, Gurusaddaiah T, Ryan CA. Proteinase inhibitor II from potatoes: isolation and characterization of its protomer components. Biochemistry. 1976 Aug 10;15(16):3418–3424. [PubMed] [Google Scholar]
- Graham JS, Pearce G, Merryweather J, Titani K, Ericsson L, Ryan CA. Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing. J Biol Chem. 1985 Jun 10;260(11):6555–6560. [PubMed] [Google Scholar]
- Graham JS, Pearce G, Merryweather J, Titani K, Ericsson LH, Ryan CA. Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II. J Biol Chem. 1985 Jun 10;260(11):6561–6564. [PubMed] [Google Scholar]
- Greenblatt HM, Ryan CA, James MN. Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from Russet Burbank potato tubers at 2.1 A resolution. J Mol Biol. 1989 Jan 5;205(1):201–228. [PubMed] [Google Scholar]
- Hass GM, Hermodson MA, Ryan CA, Gentry L. Primary structures of two low molecular weight proteinase inhibitors from potatoes. Biochemistry. 1982 Feb 16;21(4):752–756. [PubMed] [Google Scholar]
- Keil M, Sanchez-Serrano J, Schell J, Willmitzer L. Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum). Nucleic Acids Res. 1986 Jul 25;14(14):5641–5650.[PMC free article] [PubMed] [Google Scholar]
- Laskowski M, Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem. 1980;49:593–626. [PubMed] [Google Scholar]
- Melville JC, Ryan CA. Chymotrypsin inhibitor I from potatoes. Large scale preparation and characterization of its subunit components. J Biol Chem. 1972 Jun 10;247(11):3445–3453. [PubMed] [Google Scholar]
- Pearce G, Johnson S, Ryan CA. Purification and characterization from tobacco (Nicotiana tabacum) leaves of six small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family. Plant Physiol. 1993 Jun;102(2):639–644.[PMC free article] [PubMed] [Google Scholar]
- Pearce G, Sy L, Russell C, Ryan CA, Hass GM. Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases. Arch Biochem Biophys. 1982 Feb;213(2):456–462. [PubMed] [Google Scholar]
- Plunkett G, Senear DF, Zuroske G, Ryan CA. Proteinase inhibitors I and II from leaves of wounded tomato plants: purification and properties. Arch Biochem Biophys. 1982 Feb;213(2):463–472. [PubMed] [Google Scholar]
- Thornburg RW, An G, Cleveland TE, Johnson R, Ryan CA. Wound-inducible expression of a potato inhibitor II-chloramphenicol acetyltransferase gene fusion in transgenic tobacco plants. Proc Natl Acad Sci U S A. 1987 Feb;84(3):744–748.[PMC free article] [PubMed] [Google Scholar]
- Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354.[PMC free article] [PubMed] [Google Scholar]
Abstract
A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.