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

T. J. Baranski, G. Koelsch, J. A. Hartsuck, S. Kornfeld

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63 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)23365-23372
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number34
StatePublished - Dec 1 1991

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