Abstract
Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends...
Highlights
Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP- [“Clmannose into mannosylphosphorylpolyisoprenol
The [“Clmannose-labeled glycoprotein was isolated by treatment of the insoluble residue with sodium dodecyl sulfate, or water-soluble [“Clmannose-labeled glycopeptides were obtained by Pronase digestion of the insoluble material
The studies described in this paper show that cotton fibers contain enzymes which catalyze the incorporation of mannose and GlcNAc into both lipid and glycoprotein
Summary
Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP- [“Clmannose into mannosylphosphorylpolyisoprenol We show that these particulate preparations catalyze the incorporation of mannose from GDP- [‘Elmannose into lipid-linked oligosaccharides and into glycoprotein. #en p-hydroxymercuribenzoate (p-HMB) was added to incubations containing GDP- [“Clmannose and enzyme, it inhibited the formation of the oligosaccharide-lipids but allowed the particulate preparations to accumulate large amounts of [“C]mannosylphosphorylpolyisoprenol. When this inhibition was reversed by the addition of dithiothreitol, radioactivity rapidly disappeared from the mannosylphosphorylpolyisoprenol with the concomitant appearance of radioactivity in the [%]mannose-labeled oligosaccharide lipids. In the absence of GDP-mannose, essentially all of the GlcNAc is incorporated into a lipid which is soluble in CHCldCH,OH (2/l) and which was characterized as an
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