title: Isolation, affinity purification and biochemical characterization of a lysosomal cathepsin D from the deuterostome Asterias rubens.
abstract: Cathepsin D (EC 3.4.23.5) is one of the lysosomal enzymes responsible for proteolytic degradation in cells. By virtue of its mannose 6-phosphate residues, shortly after its synthesis, it is recognized by the receptors in the trans-Golgi network that mediate its transport to the lysosomes. The mammalian enzyme has been extensively characterized and several forms of cathepsin have also been identified. Cathepsins have also been isolated from other vertebrates and invertebrates and recent studies suggest that the lysosomal sorting machinery is evolutionarily conserved from fish to mammals. We recently characterized the putative mannose 6-phosphate receptors from the invertebrate starfish (Asterias rubens). In the present study we affinity purified the cathepsin D from this animal and biochemically characterized the same. Purified enzyme migrated as a single band on SDS-PAGE corresponding to a molecular mass of 45 kDa. The protein bound specifically to Con A-Sepharose gel and is glycosylated. The deglycosylated enzyme showed a molecular mass of ~40 kDa. Furthermore, an antibody raised for the purified enzyme in a rabbit recognizes the crude, the purified enzyme as well as the deglycosylated product in a western blot experiment. The enzyme in the extracts of different tissues can also be quantified by ELISA. We have further evaluated the binding of purified starfish cathepsin D with its receptor, MPR 300 (mannose 6-phosphate receptor) by immunoprecipitation. Cross-linking experiments using purified cathepsin D and MPR 300 revealed a cross-linked product that migrated with a higher molecular mass (345 kDa) compared to the enzyme (45 kDa). Furthermore the specificity of this interaction was also tested in a ligand blot experiment.
authors.affiliation: Laboratory of Protein Biochemistry and Glycobiology, Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Visa
authors.initials: V
authors.lastname: Merino
authors.forename: Nadimpalli
authors.initials: N
authors.lastname: Siva Kumar
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D047271:Asterias / Q000187:drug effects / Q000201:enzymology*; D002402:Cathepsin D / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism; D055767:Immobilized Proteins / Q000378:metabolism; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000302:isolation & purification; D047468:Immunoprecipitation; D008024:Ligands; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D003432:Cross-Linking Reagents; D006023:Glycoproteins; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D008024:Ligands; D017527:Receptor, IGF Type 2; D002402:Cathepsin D