Activation of human prolegumain by cleavage at a C-terminal asparagine residue.
The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation.
Chen
J M
JM
MRC Molecular Enzymology Laboratory, The Babraham Institute, Babraham, Cambs. CB2 4AT, U.K. jinq-may.chen@bbsrc.ac.uk
Fortunato
M
M
Barrett
A J
AJ
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D002096:C-Peptide / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099240:Asparaginyl Endopeptidase
D001216:Asparagine; D002096:C-Peptide; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D004792:Enzyme Precursors; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase