Proteolytic Processing of the gamma-Subunit Is Associated with the Failure to Form GlcNAc-1-phosphotransferase Complexes and Mannose 6-Phosphate Residues on Lysosomal Enzymes in Human Macrophages

作者：Pohl Sandra; Tiede Stephan; Marschner Katrin; Encarnacao Marisa; Castrichini Monica; Kollmann Katrin; Muschol Nicole; Ullrich Kurt; Mueller Loennies Sven; Braulke Thomas^*

来源：Journal of Biological Chemistry, 2010, 285(31): 23936-23944.

DOI：10.1074/jbc.M110.129684

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

• 出版日期2010-7-30