Characterization of the glycinin maturation protease and assembly of modified glycinin subunits
Abstract
Glycinin is the predominant 12S seed storage protein in soybean, typically accounting for about 40% of the total seed protein. It is an attractive experimental system for two reasons. First, glycinin undergoes a post-translational cleavage at a highly conserved Asn-Gly bond, a cleavage considered to be required during assembly of oligomers. It therefore serves as a model to study the relationship between protein modification and function. Second, because of its abundance, the properties of glycinin influence the nutritional properties of soy protein preparations. For example, the low sulfur amino acid content of glycinin limits the nutritional value of soy protein. Site directed mutagenesis of genes that encode glycinin is an appealing approach to solving this nutritional deficiency. To study glycinin processing, an assay was developed using a synthetic peptide that mimicked the glycinin cleavage site. This assay permitted the purification and characterization of a protease that cleaves Asn-Gly bonds. The protease cleaves synthetic proglycinin and prolegumin trimers at the Asn-Gly bond normally cleaved in vivo. It is a glycosylated sulfhydryl protease. Purified protease preparations contain three polypeptides with molecular masses of 85, 65 and 23 kDa. The 65 and 23 kDa polypeptides are dissulfide linked and are antigenically related to the 85 kDa polypeptide. Additionally, the amino terminal sequences of the 85 and 65 kDa polypeptides coincide, while the amino terminal sequence of the 23 kDa polypeptide differs. The substrate requirements of the protease were investigated by digesting 11S precursors containing modified cleavage sites. The available data are consistent with the conclusion that an Asn must precede the cleaved peptide bond but limited variation is tolerated among the two amino acids subsequent to the bond that is cleaved. To improve the nutritional characteristics of soy protein, several glycinin subunits containing elevated methionine levels were produced in vitro from modified cDNAs. The modified subunits were tested for their ability to assemble into oligomers. A hypervariable region was identified that tolerates large insertions and deletions, and certain conserved regions tolerated conservative point mutations.
Degree
Ph.D.
Advisors
Nielsen, Purdue University.
Subject Area
Biochemistry|Plant propagation
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