Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii.
The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.
Ketcham
C M
CM
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
S
S
D000048:Acanthamoeba / Q000201:enzymology*; D000135:Acid Phosphatase; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D004789:Enzyme Activation; D006031:Glycosylation; D007527:Isoenzymes; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D000071681:Tartrate-Resistant Acid Phosphatase; D017855:Transferases (Other Substituted Phosphate Groups)
D007527:Isoenzymes; D008360:Mannosephosphates; D008667:Metalloproteins; C027693:mannose-6-phosphate; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002402:Cathepsin D