Abstract
Biosynthesis of sulfated saccharides that are linked to asparagine residues in the cell surface glycoprotein of Halobacterium halobium via a glucose residue involves sulfated dolichyl-monophosphoryl oligosaccharide intermediates (Lechner, J., Wieland, F., and Sumper, M. (1985) J. Biol. Chem. 260, 860-866). During isolation and characterization of these lipid oligosaccharides we detected a group of related compounds containing additional unidentified sugar residues. Here we report that: 1) the unknown sugar residues were 3-O-methylglucose, linked peripherally to the lipid-saccharide intermediates; 2) the 3-O-methylglucose residues in the oligosaccharides occur only at the lipid-linked level but are absent at the protein-linked level; 3) cell surface glycoprotein biosynthesis in Halobacteria in vivo is drastically depressed when S-adenosylmethionine-dependent methylation is inhibited, indicating that methylation is an obligatory step during glycoprotein synthesis. We propose a mechanism for the transport of lipid oligosaccharides through the cell membrane, involving an intermediate stage in which the saccharide moieties are transiently modified with 3-O-methylglucose.
Highlights
Johann Lechner,Felix Wielandl, and Manfred Sumper From the Institut fur BiochemieI der Uniuersitat Regensburg, Uniuersitatsstrasse 31, 8400 Regensburg, Federal Republic of Germany
In thispaper we show that this residue is 3-O-methylglucose, [%]Methionine labeling
We suggest a possible function for this methylated hexose in thebiosynthesis of the halobacterial cell surface glycoprotein
Summary
0.24% (w/v) of alanine and shaken for 4 h a t 37 "C. Thereafter, the bacteria were harvested and resuspended at 20 times the original cell. Quantitative as well as qualitative sugar analyses, either by color- concentration in sulfate-free basal salts (conditions of inhibition are imetric assays or by gas liquid chromatography (GLC'),were as given in the legend to Fig. 5) and stirred under illumination at 37 "C. described in Ref. 1.Hydrolysis of uronic acid-containing saccharides After 2 h, the cells were centrifuged and resuspended in an equal was morecomplete, and.losses of material by destruction were smaller volume of sulfate-containing basalsalts (400 pl) with 150 pCiof [3H]. The abbreviations used are: GLC, gas-liquidchromatography; SP, were concentrated and chromatographed on Silica 60 thin layer plates sulfated precursor; GlcUA, glucuronic acid.
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