Activation of progelatinase A by mammalian legumain, a recently discoveredcysteine proteinase

The activation of progelatinase A to gelatinase A requires cleavage of an a sparaginyl bond to form the N-terminus of the mature enzyme. We have asked whether the activation can be mediated by legumain, the recently discovered lysosomal cysteine proteinase that is specific for hydrolysis of asparagin yl bonds. Addition of purified legumain to the concentrated conditioned med ium from HT1080 cell culture that contained both progelatinases A and B cau sed the conversion of the 72 kDa progelatinase A to the 62 kDa form. The pr ogelatinase B in the medium was unaffected. Incubation of recombinant proge latinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, Mc a-Pro-Leu-GlyLeu-Dpa-Ala-Arg-NH2. Legumain also activated progelatinase A w hen it was in complex with TIMP-2. Zymographic analysis and N-terminal sequ encing revealed that legumain cleaved the 72 kDa progelatinase A at the bon ds between Asn(109)-Tyr(110) or Asn(111)-Phe(112) to produce the 62 kDa mat ure enzyme, and that further cleavage at Asn(430) also occurred to generate a 36 kDa active form. More 62 kDa gelatinase A was detected in cultures of C13 cells that over-expressed legumain than in those of the control HEK293 cells. We conclude that legumain is clearly capable of processing progelat inase A to the active enzyme in vitro and in cultured cells.