Structural Requirements for Efficient Processing and Activation of Recombinant Human UDP-N-acetylglucosamine:Lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

• Published in: The Journal of biological chemistry 2006-04, Vol.281 (17), p.11761-11768
• Main Authors: Kudo, Mariko, Canfield, William M.
• Format: Article
• Language: English
• Subjects: alpha-Glucosidases - metabolism; Amino Acid Sequence; Animals; Cattle; CHO Cells - enzymology; Cricetinae; DNA, Complementary; Humans; Hydrolases - metabolism; Lysosomes - enzymology; Molecular Sequence Data; Phosphorylation; Protein Processing, Post-Translational; Protein Subunits; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; Transferases (Other Substituted Phosphate Groups) - chemistry; Transferases (Other Substituted Phosphate Groups) - genetics; Transferases (Other Substituted Phosphate Groups) - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M513717200

Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an α2β2γ2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the α- and β-subunits are encoded by a single gene as a precursor, whereas the γ-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the α- and β-subunits but not the γ-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the α- and β-subunits. Because this modification prevented precursor processing to mature α- and β-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified α/β-subunits (α′/β′-subunits) precursor and wild type γ-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an α′2β′2γ2-subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAc-phosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid α-glucosidase in vitro.