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

Abstract

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, ␣ 2 ␤ 2 ␥ 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 ␥-subunit but not the ␤-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 ␥-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the ␥-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

Post-translational Modifications of the γ-Subunit Affect Intracellular Trafficking and Complex Assembly of GlcNAc-1-phosphotransferase

“…N-Glycosylation Is Required for the Intracellular Trafficking of the ␥-Subunit-The endogenous human ␥-subunit of GlcNAc-1-phosphotransferase is localized in the cis-Golgi apparatus in human fibroblasts and cultured macrophages (12,13,18). Double immunofluorescence microscopy of mouse embryonic fibroblasts demonstrated the presence of the endogenous ␥-subunit in the cis-Golgi apparatus by complete colocalization with the Golgi apparatus marker GM130 (supplemental Fig.…”

“…Size exclusion chromatography represents a suitable method to follow the assembly of the GlcNAc-1-phosphotransferase subunits into higher molecular mass complexes (13). Here, we analyzed the importance of N-linked oligosaccharides and the capability of the ␥-subunit for dimerization to assemble in high molecular mass GlcNAc-1-phosphotransferase complexes.…”

“…For enzymatic deglycosylation of proteins, cell extracts were incubated in the presence or absence of PNGase F, endoglycosidase H, or sialidase for 1-3 h at 37°C according to the manufacturer's instructions. Gel filtration chromatography of extracts of COS-7 cells overexpressing ␥-subunit constructs was performed in the presence of 30 mM octyl-␤-D-glucopyranoside on a SMART HPLC system as described recently (13).…”

“…The ␥-subunit of GlcNAc-1-phosphotransferase represents a soluble glycoprotein of 305 amino acids that forms disulfidelinked homodimers (5). It is retained in the lumen of the cisGolgi apparatus, most likely by interactions with the integral ␣-and ␤-subunits of the GlcNAc-1-phosphotransferase complex (12,13). The role of ␥-subunits in the formation of Man-6-P residues is not yet clear.…”

“…From recent studies, it has been concluded that the ␥-subunit is important either to facilitate the proper folding of the subunits of GlcNAc-1-phosphotransferase and to maintain them in a conformation competent for substrate recognition and binding or to regulate the activity and expression of the ␣/␤-subunits (9,14,15). Furthermore, it was shown that proteolytic fragmentation of the ␥-subunit in human macrophages is associated with reduced activity of the GlcNAc-1-phosphotransferase complex (13).…”