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.
Highlights
We have investigated the natuorfea protein domain soma1 compartment where the hydrolases are subsequently that is shared among lysosomal hydrolases andis rec- packaged into lysosomes
230, lysine 203, and 265-292) from the carboxyl lobe of the lysosomal hydrolase cathepsin D into thehomologous positions of the related secretory protein glycopepsinogen was sufficient to confer recacetylglucosamine 1-phosphate to mannose residues on lysosomal enzymes to form phosphodiester intermediates
The current study demonstrates thatwhen cally phosphorylates the high mannose oligosaccharides of these two regions are replaced in cathepsin D by the the lysosomal enzymes [3,4,5]
Summary
Density (1.75 ml of 0.4 mg receptor/ml resin) MPR-Affi-Gel affinity columns were used for the initial experiments and were later replaced. Materials-The preparation of rabbit antiserato human cathepsin by high density (1.5 ml of 2.2 mg receptor/ml resin) MPR-Affi-Prep. Fractions corresponding to two oocyteequivalents receptor (MPR) was prepared as described previously [13].Mannose- of column run-through and Man-6-P eluted material were immuno-. DNA polymerase were purchased from Pharmacia LKB Biotechnol- umns bound up to 25% more of the expressed proteins compared with ogy Inc. Geneclean Kit was purchased from BIO 101, Inc. Endo-@- the binding to the“low density” columns. EXPRE35S35S (L-methionine, [35S]:L-cysteine, [35S]),>lo00 Ci/mmol and GDP[3,4-3H]mannose, Ci/mmol, were purchased from Du Pont-
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