Does an asparaginyl-specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings?

An asparaginyl-specific cysteine endopeptidase which was named 'legumain-li ke proteinase' (LLP) and has an apparent molecular mass of 38.1 kDa was iso lated from cotyledons of kidney bean (Phaseolus vulgaris L.) seedlings and partially characterized. It is, to our knowledge, the first known proteinas e which in vitro extensively degrades native phaseolin, the major storage g lobulin of this grain legume. Phaseolin that in vitro had been partially degraded by LLP (Pvitro) and pha seolin that was isolated after partial in vivo breakdown 6 days after the s tart of seed imbibition (Pvivo) showed similar fragment patterns on SDS/pol yacrylamide gels. The fragments had identical cleavage sites in Pvitro and Pvivo as determined by partial amino acid sequencing. In both types of part ially degraded phaseolin, these cleavage sites have asparagine in the P1 po sition. Two of the cleavage sites are located in the beta-barrel domain of the C-te rminal module and only one cleavage site was found in the beta-barrel domai n of the N-terminal module according to the consensus structural model of p haseolin subunits. These results suggest that very likely LLP could in vivo be responsible for the initiation of phaseolin proteolysis. Two different legumain-specific clones named cp6b and p21b were isolated from a cDNA libr ary of germinated bean cotyledons. Cp6b encodes LLP, while p21b encodes a V PE-like enzyme. Southern-blot analysis revealed a single gene copy for PV-V PE and, presumably, at least two gene copies for LLP in the kidney bean gen ome. Northern-blot analysis indicated that mRNAs for both clones appear de novo during seed germination. However, the developmental patterns of the tr anscript levels corresponding to the two clones differed significantly. The temporal pattern of phaseolin degradation and of LLP polypeptide levels ag reed well with the suggestion that LLP plays a key role in the mobilization of phaseolin during and after kidney bean germination.