Proteolytic Characterization and Lysosomal Localization of Echinoderm Cathepsin D

Abstract

Cathepsin D is an important lysosomal aspartic protease and is transported to the lysosomes in both mannose 6-phosphate dependent and independent manner. The present study reports lysosomal transport and proteolytic characterization of cathepsin D purified from gonads of starfish Asterias Rubens. Activity of the purified enzyme was confirmed by a zymogram assay on hemoglobin. Characterization of proteolytic activity on bovine serum albumin and ovalbumin showed that the enzyme cleaves these proteins with high specificity when compared to human enzymes. Lysosomal localization of the purified enzyme in human embryonic kidney cells was studied using Fluorescein isothiocyanate conjugated enzyme and anti-goat cation independent mannose 6-phosphate receptor antibody. Primary sequence based studies and phylogenetic analysis showed that the echinoderm cathepsin D is more related to plant aspartic protease phytepsin and may have three intra molecular disulfide linkages. The results show that the lysosomal transport of the enzyme in marine invertebrates is facilitated through the mannose 6-phosphate (m6-p) receptor mediated pathway even though m6-p independent pathways for homologues of the enzyme are also prevalent in higher organisms.