Lysosomal enzyme phosphorylation. I. Protein recognition determinants in both lobes of procathepsin D mediate its interaction with UDP- GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase

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Abstract

We have investigated the nature of a protein domain that is shared among lysosomal hydrolases and is recognized by UDP-GlcNAc:lysosomal enzyme N- acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues. Previously, elements of this recognition domain were identified using a chimeric protein approach. The combined substitution of two regions (amino acids 188-230, particularly lysine 203, and 265-292) from the carboxyl lobe of the lysosomal hydrolase cathepsin D into the homologous positions of the related secretory protein glycopepsinogen was sufficient to confer recognition by phosphotransferase and subsequent phosphorylation of the oligosaccharides when this chimeric protein was expressed in Xenopus oocytes. (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). The current study demonstrates that when these two regions are replaced in cathepsin D by the homologous glycopepsinogen amino acids, the resultant chimeric molecule is poorly phosphorylated. However, when either of these regions is substituted individually, the chimeric molecules are well phosphorylated. The phosphorylation of these latter chimeric proteins is dependent on the presence of procathepsin D amino lobe elements. By analyzing a series of chimeric proteins that contain all eight combinations of three consecutive segments of the entire amino lobe of procathepsin D, it was found that multiple regions of the amino lobe of cathepsin D enhance phosphorylation of the chimeric proteins. These elements may be part of an extended carboxyl lobe recognition domain or comprise a second independent recognition domain.

Original languageEnglish
Pages (from-to)23342-23348
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number32
StatePublished - Jan 1 1992

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