Previous studies have shown that lysine residues on the surface of cathepsins and other lysosomal proteins are a shared component of the recognition structure involved in mannose phosphorylation. In this study, the involvement of specific lysine residues in mannose phosphorylation of cathepsin D was explored by site-directed mutagenesis. Mutation of two lysine residues in the mature portion of the protein, Lys-203 and 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 explained by alterations in protein folding. Structural comparisons of the two lysine residues with those required for phosphorylation of cathepsin L, using models generated from recently acquired crystal structures, revealed several relevant similarities. On both molecules, the lysine residues were positioned approximately 34 Å apart (34.06 Å for cathepsin D and 33.80 Å for cathepsin L). When the lysine pairs were superimposed, N-linked glycosylation sites on the two proteins were found to be oriented so that oligosaccharides extending out from the sites could share a common region of space. Further similarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeting structure based on a specific arrangement of lysine residues with respect to each other and to glycosylation sites on the surface of lysosomal proteins.The mammalian lysosomal protein targeting system has the capability of recognizing and modifying lysosomal hydrolases and growth factors from a wide range of protein families with high specificity. The molecular basis for this selectivity is due to the activity of UDP-GlcNAc, lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, which phosphorylates Nlinked oligosaccharides of lysosomal proteins by the addition of phospho-GlcNAc (1-5). This modification begins after lysosomal proteins are exported from the endoplasmic reticulum and is followed by removal of terminal GlcNAc moieties and binding to mannose 6-phosphate receptors as the proteins traverse the Golgi apparatus. The receptors mediate delivery of the phosphorylated proteins to the endosomal compartment, and from there the proteins are transported to lysosomes (for a review, see Ref. 1).Studies focusing on the molecular basis for mannose phosphorylation have shown that the phosphotransferase recognizes a protein-based structure or "signal" on lysosomal proteins and that this recognition is required for efficient phosphorylation in vivo and in vitro (5). Expression of this phosphorylation signal requires that the protein be in its native conformation (4 -6). Analysis of the cathepsin D signal, using chimeras of cathepsin D and the nonphosphorylated homologous aspartic protease pepsinogen, defined a "minimal" structure for phosphorylation involving Lys-203 and a segment of 27 ami...
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