Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP)

A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyled ons of germinated Vigna mungo seeds and is responsible for degradation of t he seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal pr osegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endope ptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-termin al post-translational processing of SH-EP. The recombinant preform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity a nd refolded by stepwise dialysis. P-31-NMR analysis of intact germinated co tyledons revealed that the vacuolar pH of cotyledonary cells changes from 6 .04 to 5.47 during seed germination and early seedling growth. rSH-EP was c onverted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination , but not at the pH of the early stage. VmPE-1 accelerated the rate of proc essing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the ea rly and mid stages of germination. In addition, the cleavage sites of the i n vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We prop ose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germinat ion, and both VmPE-1-mediated and autocatalytic processings function synerg istically in the activation of proSH-EP in cotyledons at the mid and late s tages.