Direct affinity purification and supramolecular organization of human lysosomal cathepsin A.
Cathepsin A (also named "protective protein" and carboxypeptidase L) stabilizes beta-galactosidase and activates neuraminidase by forming with them a high-molecular-weight lysosomal complex. We determined the main forms of the supramolecular organization of human placental cathepsin A and the quantitative relationship between them, using an affinity chromatography on agarose-Phe-Leu for direct purification of cathepsin A. We found that cathepsin A in human placenta exists as the following three forms: a 1270-kDa complex with beta-galactosidase and neuraminidase (about 1% of total cathepsin A), a 680-kDa complex with beta-galactosidase (30-40% of total), and a free 98-kDa cathepsin A dimer (60-70% of total). All forms are in dynamic equilibrium with each other, but almost all placental beta-galactosidase is associated with cathepsin A in the 680-kDa complex. The main properties of free cathepsin A (including the capacity to associate with beta-galactosidase) were found to be identical to those of cathepsin A obtained by dissociation of the 680-kDa complex. The presence of a free cathepsin A pool in the lysosome is connected with its sixfold overproduction in the cell compared to beta-galactosidase and may be necessary to ensure cathepsin A proteolytic function in addition to its protective role for beta-galactosidase and neuraminidase in the lysosomal multienzymatic complex. Such a dual function of cathepsin A is also confirmed by our finding that it is the only carboxypeptidase of placenta extract able to catalyze the hydrolysis of both carbobenzoxy (CBZ)-Glu-Tyr and CBZ-Phe-Leu dipeptide substrates.
Pshezhetsky
A V
AV
Service de Génétique Médicale, Hôpital Sainte-Justine, Montréal, Québec, Canada.
Potier
M
M
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000737:chemistry*; D043402:Cathepsin A; D002403:Cathepsins / Q000737:chemistry*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology / Q000648:ultrastructure*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry; D010920:Placenta / Q000201:enzymology / Q000648:ultrastructure; D011485:Protein Binding; D001616:beta-Galactosidase / Q000737:chemistry
D046911:Macromolecular Substances; D009439:Neuraminidase; D001616:beta-Galactosidase; D002268:Carboxypeptidases; D002403:Cathepsins; C468629:CTSA protein, human; D043402:Cathepsin A