Structural requirements for efficient processing and activation of recombinant human UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase.
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 alpha2beta2gamma2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the alpha- and beta-subunits are encoded by a single gene as a precursor, whereas the gamma-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the alpha- and beta-subunits but not the gamma-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 alpha- and beta-subunits. Because this modification prevented precursor processing to mature alpha- and beta-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified alpha/beta-subunits (alpha'/beta'-subunits) precursor and wild type gamma-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 alpha'2beta'2gamma2-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 alpha-glucosidase in vitro.
Kudo
Mariko
M
Genzyme Corporation, Oklahoma City, Oklahoma 73104, USA.
Canfield
William M
WM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000201:enzymology; D002417:Cattle; D006224:Cricetinae; D018076:DNA, Complementary; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism
D018076:DNA, Complementary; D021122:Protein Subunits; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D006867:Hydrolases; D000520:alpha-Glucosidases