Lysosomal protective protein/cathepsin A. Role of the "linker" domain in catalytic activation.
Lysosomal protective protein/cathepsin A is a serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. The enzyme is synthesized as a 54-kDa precursor/zymogen and processed into a catalytically active 32- and 20-kDa two-chain form. We have expressed in baculovirus-infected insect cells the human one-chain precursor as well as the two separate subunits in order to establish the mode of catalytic activation of the zymogen and the assembly and activation of the two subunits. Infected insect cells synthesize large quantities of the exogenous proteins, which are glycosylated and secreted but not processed. Co-expression of the two subunits results in their assembly into a two-chain form of 34- and 20-kDa with negligible enzymatic activity. Limited proteolysis with trypsin of the 54-kDa precursor and the reconstituted 34- and 20-kDa form gives rise to a fully active 32- and 20-kDa product. These results enabled us to map the sites of proteolytic cleavage needed for full activation of the cathepsin A zymogen. They further indicate that the 34- and 20-kDa form is a transient processing intermediate that is converted into a mature and active enzyme by removal of a 2-kDa "linker" peptide from the COOH terminus of the 34-kDa subunit.
Bonten
E J
EJ
Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Galjart
N J
NJ
Willemsen
R
R
Usmany
M
M
Vlak
J M
JM
d'Azzo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae; D002268:Carboxypeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D043402:Cathepsin A; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure; D014162:Transfection
D000906:Antibodies; D004792:Enzyme Precursors; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D004229:Dithiothreitol