Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting

• Published in: The Journal of biological chemistry 1991-12, Vol.266 (34), p.23365-23372
• Main Authors: BARANSKI, T. J, KOELSCH, G, HARTSUCK, J. A, KORNFELD, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Biological and medical sciences; Cathepsin D - chemistry; Cathepsin D - metabolism; Cell structures and functions; Computer Simulation; Fundamental and applied biological sciences. Psychology; Humans; Lysosome, phagosome; Lysosomes - enzymology; Models, Molecular; Molecular and cellular biology; Molecular Sequence Data; Mutagenesis; Phosphotransferases - metabolism; Sequence Alignment; site-directed mutagenesis; Swine; Transferases (Other Substituted Phosphate Groups)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54505-1

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose-6-P residues. Previously, we generated a lysosomal enzyme recognition domain by substituting two regions (lysine 203 and amino acids 265-292) of the lysosomal hydrolase cathepsin D into a related secretory protein glycopepsinogen. When expressed in Xenopus oocytes, the oligosaccharides of the chimeric protein were efficiently phosphorylated (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). In the current study, incremental substitutions of cathepsin D residues into glycopepsinogen and alanine-scanning mutagenesis were utilized to define the recognition domain more precisely. A computer-generated model of the cathepsin D/pepsinogen chimeric molecule served as a guide for mutagenesis and for the interpretation of results. These studies indicate that the recognition domain is a surface patch that contains multiple interacting sites. There is a strict positional requirement for the lysine residue at position 203.