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.
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 GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.
Pohl
Sandra
S
Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
Tiede
Stephan
S
Marschner
Katrin
K
Encarnação
Marisa
M
Castrichini
Monica
M
Kollmann
Katrin
K
Muschol
Nicole
N
Ullrich
Kurt
K
Müller-Loennies
Sven
S
Braulke
Thomas
T
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002402:Cathepsin D / Q000737:chemistry; D002522:Chlorocebus aethiops; D002845:Chromatography / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008264:Macrophages / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D008360:Mannosephosphates / Q000737:chemistry*; D008954:Models, Biological; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics* / Q000502:physiology
D008360:Mannosephosphates; C027693:mannose-6-phosphate; D017855:Transferases (Other Substituted Phosphate Groups); C511050:GNPTAB protein, human; D002402:Cathepsin D