Activation of progelatinase A by mammalian legumain, a recently discovered cysteine proteinase.
The activation of progelatinase A to gelatinase A requires cleavage of an asparaginyl 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 asparaginyl bonds. Addition of purified legumain to the concentrated conditioned medium from HT1080 cell culture that contained both progelatinases A and B caused the conversion of the 72 kDa progelatinase A to the 62 kDa form. The progelatinase B in the medium was unaffected. Incubation of recombinant progelatinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Legumain also activated progelatinase A when it was in complex with TIMP-2. Zymographic analysis and N-terminal sequencing revealed that legumain cleaved the 72 kDa progelatinase A at the bonds between Asn109-Tyr110 or Asn111-Phe112 to produce the 62 kDa mature enzyme, and that further cleavage at Asn430 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 progelatinase A to the active enzyme in vitro and in cultured cells.
Chen
J M
JM
MRC Molecular Enzymology Laboratory, The Babraham Institute, Cambridgeshire, UK.
Fortunato
M
M
Stevens
R A
RA
Barrett
A J
AJ
D000595:Amino Acid Sequence; D002460:Cell Line; D017077:Culture Media, Conditioned; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018093:Gelatinases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D000099240:Asparaginyl Endopeptidase
D017077:Culture Media, Conditioned; D003546:Cysteine Endopeptidases; D004792:Enzyme Precursors; D018093:Gelatinases; D008666:Metalloendopeptidases; D010940:Plant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D000099240:Asparaginyl Endopeptidase; C069860:progelatinase