Characterization of UDP-N-acetylglucosamine:glycoprotein N- acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii

C. M. Ketcham, S. Kornfeld

Research output: Contribution to journalArticle

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Abstract

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 (K(i) of 48 μM) but did not inhibit the phosphorylation of RNase B or the simple sugar α-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 α1,2-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 α-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.

Original languageEnglish
Pages (from-to)11654-11659
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number16
StatePublished - Jan 1 1992

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