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. 1993 Apr;101(4):1341–1348. doi: 10.1104/pp.101.4.1341

Activation of bean (Phaseolus vulgaris) alpha-amylase inhibitor requires proteolytic processing of the proprotein.

J J Pueyo 1, D C Hunt 1, M J Chrispeels 1
PMCID: PMC160658  PMID: 8310064

Abstract

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the alpha-amylases of mammals and insects. This alpha-amylase inhibitor (alpha AI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M(r)) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to alpha AI recognize large (M(r) 30,000-35,000) polypeptides as well as typical alpha AI processing products (M(r) 15,000-18,000). Alpha AI activity was found in all extracts that had the typical alpha AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant alpha AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-alpha AI when the gene is expressed in tobacco. When pro-alpha AI was separated from mature alpha AI by gel filtration, pro-alpha AI was found not to have alpha-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.

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Selected References

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  1. Altabella T., Chrispeels M. J. Tobacco Plants Transformed with the Bean alphaai Gene Express an Inhibitor of Insect alpha-Amylase in Their Seeds. Plant Physiol. 1990 Jun;93(2):805–810. doi: 10.1104/pp.93.2.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chrispeels M. J., Higgins T. J., Spencer D. Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. J Cell Biol. 1982 May;93(2):306–313. doi: 10.1083/jcb.93.2.306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dickinson C. D., Hussein E. H., Nielsen N. C. Role of posttranslational cleavage in glycinin assembly. Plant Cell. 1989 Apr;1(4):459–469. doi: 10.1105/tpc.1.4.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Graham J. S., Pearce G., Merryweather J., Titani K., Ericsson L., Ryan C. A. 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]
  5. Hara-Nishimura I., Inoue K., Nishimura M. A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett. 1991 Dec 2;294(1-2):89–93. doi: 10.1016/0014-5793(91)81349-d. [DOI] [PubMed] [Google Scholar]
  6. Julius D., Brake A., Blair L., Kunisawa R., Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell. 1984 Jul;37(3):1075–1089. doi: 10.1016/0092-8674(84)90442-2. [DOI] [PubMed] [Google Scholar]
  7. Klionsky D. J., Herman P. K., Emr S. D. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. doi: 10.1128/mr.54.3.266-292.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Marshall J. J., Lauda C. M. Purification and properties of phaseolamin, an inhibitor of alpha-amylase, from the kidney bean, Phaseolus vulgaris. J Biol Chem. 1975 Oct 25;250(20):8030–8037. [PubMed] [Google Scholar]
  10. Moreno J., Altabella T., Chrispeels M. J. Characterization of alpha-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris. Plant Physiol. 1990 Mar;92(3):703–709. doi: 10.1104/pp.92.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Neurath H. The versatility of proteolytic enzymes. J Cell Biochem. 1986;32(1):35–49. doi: 10.1002/jcb.240320105. [DOI] [PubMed] [Google Scholar]
  12. Osborni T. C., Alexander D. C., Sun S. S., Cardona C., Bliss F. A. Insecticidal activity and lectin homology of arcelin seed protein. Science. 1988 Apr 8;240(4849):207–210. doi: 10.1126/science.240.4849.207. [DOI] [PubMed] [Google Scholar]
  13. Pick K. H., Wöber G. Proteinaceous alpha-amylase inhibitor from beans (Phaseolus vulgaris). Purification and partial characterization. Hoppe Seylers Z Physiol Chem. 1978 Oct;359(10):1371–1377. doi: 10.1515/bchm2.1978.359.2.1371. [DOI] [PubMed] [Google Scholar]
  14. Scott M. P., Jung R., Muntz K., Nielsen N. C. A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):658–662. doi: 10.1073/pnas.89.2.658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sharon N., Lis H. Legume lectins--a large family of homologous proteins. FASEB J. 1990 Nov;4(14):3198–3208. doi: 10.1096/fasebj.4.14.2227211. [DOI] [PubMed] [Google Scholar]
  16. Wilcox E. R., Whitaker J. R. Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase. Biochemistry. 1984 Apr 10;23(8):1783–1791. doi: 10.1021/bi00303a031. [DOI] [PubMed] [Google Scholar]
  17. Yamaguchi H. Isolation and characterization of the subunits of Phaseolus vulgaris alpha-amylase inhibitor. J Biochem. 1991 Nov;110(5):785–789. doi: 10.1093/oxfordjournals.jbchem.a123660. [DOI] [PubMed] [Google Scholar]

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