Addition of glucose to dolichyl diphosphate oligosaccharide and transfer to protein.
TL;DR: The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose, but here the compound with three glucoses was used as donor in the transfer to protein.
Abstract: The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose. Paper chromatography allowed the separation of oligosaccharides (obtained by acid hydrolysis of the dolichyl diphosphate derivative) containing 1, 2 and 3 glucose residues. Using this procedure it was found that the addition of all three glucoses to the dolichyl diphosphate oligosaccharide occur with dolichyl phosphate glucose as donor. Furthermore only the compound with three glucoses was used as donor in the transfer to protein. The addition of glucose to exogenous dolichyldiphosphate oligosaccharide labelled by transfer from radioactive guanosine diphosphate mannose was detected.
Summary (1 min read)
MATERIALS AND METHODS
- Rat liver microsomes were prepared as described previously  .
- Dol-P was isolated and purified up to the DEAE-cellulose step  .
- Dol-P-P-Glc-oligosaccharide labelled in the glucose or in the mannose was obtained by incubation of microsomes with UDP-['4C]G1~ .
- The glucose-containing oligosaccharides with three, two and one glucose residues are referred to as Glc3, Glcz and Glcl oligosaccharides.
- A similar preparation has now been found to contain four substances when analyzed under the new conditions (Fig. 3 ).
- Although it was shown some time ago  that Dol-P-Glc can serve as donor for the formation of Dol-P-P oligosaccharide it had not been established whether all the glucoses are transferred by that mechanism.
- To decide this point, labelled Dol-P-Glc was incubated with liver microsomes and the Dol-P-P-Glc-oligosaccharide formed was hydrolysed and the oligosaccharides were chromatographed as described before.
- Presumably there occurs a competition between UDP-Glc and GDP-Man for the Dol-P available.
- The solvent for paper chromatography described in this paper is nearly the same as the one used by Huber et al.  for thin-layer chromatography.
- The neat separation of the different glucose-containing oligosaccharides has led to the clarification of several details of their transformations.
- Their rate of transfer is slower and may involve endogenous glucose donors.
- The formation of Dol-P-P-Glc oligosaccharide has been carried out before by transfer from Dol-P-Glc to an endogenous acceptor present in the microsomal preparations  .
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