Inhibition of glycosylation by amphomycin and sugar nucleotide analogs PP36 and PP55 indicates that Haloferax volcanii beta-glucosylates both glycoproteins and glycolipids through lipid-linked sugar intermediates: evidence for three novel glycoproteins and a novel sulfated dihexosyl-archaeol glycolipid.
Arachaebacteria have been recently placed in evolution as a separate kingdom of organisms between procaryotes and eucaryotes. Although these organisms contain both glycolipids and glycoproteins, they possess no Golgi. No biosynthetic work has been published on the complex carbohydrates of these newly reassigned organisms. This report describes preliminary results from one member of this kingdom, Haloferax volcanii, which suggest that all glycosylation proceeds through lipid intermediates. Evidence for novel glycolipid structure was also found during this study. H. volcanii plasma membranes contain all of the enzyme activities for synthesis of N-linked glycoproteins and archaeol-based glycolipids. For glucose transfer, all reactions apparently proceed through glucose-phosphopolyisoprenol using UDP-glucose as primary donor. Incorporation of D-[3H]glucose from UDP-D-[3H]glucose into glycoproteins and glycolipids of H. volcanii was stimulated by addition of C55-polyisoprenol phosphate, but not by C85-105 dolichol phosphate, and was inhibited by amphomycin and two recently described sugar nucleotide analogs, PP36 (5'-[N-(2-decanoylamino-3-hydroxy-3-phenylpropyloxy carbonyl)glycyl]amino]-5'-deoxyuridine) and PP55 (5'-O-[[(2-decanoylamino-3-phenylpropyloxycarbonyl) amino]sulfonyl]uridine). All three inhibitors are reported to block transfer of sugar from UDP-sugars to phosphopolyisoprenols in eucaryotes. However, in H. volcanii these inhibitors apparently block transfer of glucose from polyprenyl intermediates to final glycoproteins and glycolipid products. The sulfodihexosyl archaeol glycolipid fraction was partially characterized by mass spectrometry and was found to contain a previously unreported structure with sulfate on the reducing-end sugar. Four major glycoproteins 190, 105, 56, and 52 kDa and an archaeol-based glycolipid fraction were labeled by amphomycin-sensitive pathways. Photoaffinity labeling of H. volcanii homogenate with 5-azido-[32P]UDP-Glc tagged only one 45-kDa polypeptide which is a probable glucosyl-phosphorylpolyisoprenol synthase. The fact that only one polypeptide band was photoaffinity-labeled indicated that no other transferase utilized UDP-glucose directly in H. volcanii. The salt requirement of the UDP-glucose-dependent pathways suggests that cytoplasmic enzymes function in a high salt environment in H. volcanii. The archaebacterial plasma membrane thus expresses many functions for glycosylation of both glycoproteins and glycolipids, normally found in the endoplasmic reticulum and Golgi of eucaryotes.
Zhu
B C
BC
Department of Biochemistry, Louisiana State University, Baton Rouge 70803, USA.
Drake
R R
RR
Schweingruber
H
H
Laine
R A
RA
D001105:Archaea / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006017:Glycolipids / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D055666:Lipopeptides; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011412:Propanolamines / Q000378:metabolism / Q000494:pharmacology*; D013450:Sulfones / Q000378:metabolism / Q000494:pharmacology*; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D006017:Glycolipids; D006023:Glycoproteins; D055666:Lipopeptides; D009842:Oligopeptides; D011412:Propanolamines; D013450:Sulfones; C081648:5'-((N-(2-decanoylamino-3-hydroxy-3-phenylpropyloxycarbonyl)glycyl)amino)-5'-deoxyuridine; C081650:5'-O-(((2-decanoylamino-3-phenylpropyloxycarbonyl)amino)sulfonyl)uridine; C004423:amphomycin; D014532:Uridine Diphosphate Glucose; D003857:Deoxyuridine; D014529:Uridine