Degradation of ß-conglycinin ß-homotrimer by papain: independent occurrence of limited and extensive proteolysis.

Limited and extensive proteolysis occur when ß-conglycinin ß homo-trimer (ß(3)-conglycinin) from soybeans is attacked by papain. Slow limited proteolysis is restricted to cleavage of ß(3)-conglycinin polypeptides into subunit halves (N- and C-terminal domains) that are further slightly truncated. The kinetics of limited and extensive proteolyses analyzed separately indicates that the two processes occur independently from the very beginning of the reaction. In contrast, limited proteolysis of phaseolin from common beans has been found to be prerequisite for the onset of its extensive proteolysis. The dramatic distinction between the degradation patterns of ß(3)-conglycinin and phaseolin, homologous storage 7S globulins, suggests the existence of intrinsic differences in their structures. This hypothesis is supported by comparative analysis of the accessibilities to the solvent of amino acid residues in phaseolin and ß(3)-conglycinin structures, which indicated the relatively low packing density of the latter, resulting in enhanced susceptibility of it to extensive proteolysis.

New aspects of the molecular evolution of legumains, Asn-specific cysteine proteinases.

The molecular evolution of asparagine-specific cysteine proteinases, called legumains, from plants and animals was analyzed using newly available related amino acid sequences from lower eukaryotes, bacteria and Archaea. The results suggest that genuine legumains originate from prokaryote pro-legumains. The evolutionary roots of genuine legumains from plants and animals descend from Parabasalia and Alveolata before developing into their respective separate branches headed by Chlorophyta and Placozoa. The branch of legumain-like plant/animal glycosylphosphatidyl inositol transamidases separated from the general evolutionary stem of legumains at the level of lower eukaryotes. Modeling of the 3D structure of a plant genuine legumain underlined the previously suggested similarity of the active site geometry of legumains with caspases, which are Asp-specific bacterial and eukaryote proteinases.

Soybean basic 7S globulin: subunit heterogeneity and molecular evolution.

Basic 7S globulin, a cysteine-rich protein from soybean seeds, consists of subunits containing 27 kD and 16 kD chains linked by disulfide bonding. Three differently sized subunits of the basic 7S globulin were detected and partially separated by SP Sepharose chromatography. The basic 7S globulin was characterized as a member of a superfamily of structurally related but functionally distinct proteins descended from a specific group of plant aspartic proteinases.

Crystal structures and structural stabilities of the disulfide bond-deficient soybean proglycinin mutants C12G and C88S.

The constituent subunits of seed storage protein 11S globulin have two disulfide bonds that are common among 11S globulins from legume and nonlegume seeds. In the case of the A1aB1b subunit of soybean 11S globulin, glycinin, Cys12-Cys45 and Cys88-Cys298 are observed by X-ray crystallography. The significance of these two disulfide bonds for structural stability was investigated by mutagenesis of Cys12 to Gly and of Cys88 to Ser. The disulfide bond-deficient mutants C12G and C88S could form the correct conformations identical to that of the wild-type proglycinin except in the vicinities of the mutation sites C12 and C88 as shown by their crystal structures. Thermal stability monitored by differential scanning calorimetry of the mutants indicated that the contribution of these disulfide bonds to the thermal stability of proglycinin A1aB1b is low, although there is a small difference in the extent of the contribution between the two disulfide bonds (Cys12-Cys45 > Cys88-Cys298). The contribution of Cys88-Cys298 to the resistance of proglycinin A1aB1b to proteinase digestion is higher than that of Cys12-Cys45. Possible effects of structure on the different properties of C12G and C88S are discussed.

Legumains and their functions in plants.

Legumains are a family of plant and animal Asn-specific cysteine proteinases with extra-cytoplasmic localization in vacuoles or cell walls. Plant legumains are involved in Asn-specific propolypeptide processing during, for example, storage-protein deposition in maturing seeds, when these proteins are resistant against degradation by legumains. With the transition to germination and subsequent seedling growth, storage proteins are opened to unlimited cleavage by legumains, which now contribute to protein mobilization. Here, we suggest a hypothesis that unifies both functions of legumains. Their action as propolypeptide-processing or protein-degrading enzymes is naturally controlled by the conformational state of their substrates, which undergo development- or environment-dependent changes. The suggested substrate conformation-dependent differential roles of legumains might not be restricted to seeds but could also apply to cells of different tissues in vegetative organs.