LYSINE-BASED STRUCTURE RESPONSIBLE FOR SELECTIVE MANNOSE PHOSPHORYLATION OF CATHEPSIN-D AND CATHEPSIN-L DEFINES A COMMON STRUCTURAL MOTIF FOR LYSOSOMAL-ENZYME TARGETING

Previous studies have shown that lysine residues on the surface of cat hepsins and other lysosomal proteins are a shared component of the rec ognition structure involved in mannose phosphorylation, In this study, the involvement of specific lysine residues in mannose phosphorylatio n of cathepsin D was explored by site-directed mutagenesis, Mutation o f two lysine residues in the mature portion of the protein, Lys-203 an d Lys-293, cooperated to inhibit mannose phosphorylation by 70%. Other positively charged residues could not substitute for lysine at these positions, and comparison of thermal denaturation curves for the wild type and mutant proteins indicated that the inhibition could not be ex plained by alterations in protein folding. Structural comparisons of t he two lysine residues with those required for phosphorylation of cath epsin L, using models generated from recently acquired crystal structu res, revealed several relevant similarities. On both molecules, the ly sine residues were positioned approximately 34 Angstrom apart (34.06 A ngstrom for cathepsin D and 33.80 Angstrom for cathepsin L), When the lysine pairs were superimposed, N-linked glycosylation sites on the tw o proteins were found to be oriented so that oligosaccharides extendin g out from the sites could share a common region of space. Further sim ilarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeti ng structure based on a specific arrangement of lysine residues with r espect to each other and to glycosylation sites on the surface of lyso somal proteins.