Human lysosomal beta-galactosidase-cathepsin A complex: definition of the beta-galactosidase-binding interface on cathepsin A.
Human lysosomal beta-galactosidase is organized as a 680-kDa complex with cathepsin A (also named carboxypeptidase L and protective protein), which is necessary to protect beta-galactosidase from intralysosomal proteolysis. To understand the molecular mechanism of beta-galactosidase protection by cathepsin A, we defined the structural organization of their complex including the beta-galactosidase-binding interface on cathepsin A. Radiation inactivation analysis suggested the existence of a 168-kDa structural subunit of the complex containing both beta-galactosidase and cathepsin A. Chemical cross-linking of the complex confirmed the existence of this subunit and showed that it is composed of one cathepsin A dimer and one beta-galactosidase monomer. The modeling of the cathepsin A dimer tertiary structure based on atomic coordinates of a wheat carboxypeptidase suggested a putative beta-galactosidase-binding cavity formed by the association of two cathepsin A monomers. According to this model two exposed loops of cathepsin A bordering the cavity were chosen as part of a putative beta-galactosidase-binding interface. Synthetic peptides corresponding to these loops were found both to dissociate the complex and to inhibit its in vitro reconstitution from purified cathepsin A and beta-galactosidase. The defined location of the GAL monomer in the complex with 35% of its surface covered by the CathA dimer may explain the stabilizing effect of CathA on GAL in lysosome.
Pshezhetsky
A V
AV
Service de Génétique Médicale, Hôpital Sainte-Justine, Montréal, Québec, Canada.
Elsliger
M A
MA
Vinogradova
M V
MV
Potier
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D043402:Cathepsin A; D003432:Cross-Linking Reagents; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D001616:beta-Galactosidase; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A