Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and 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.There is evidence showing that the donor for the glycosylation of asparagine residues in proteins is a dolichyl diphosphate oligosaccharide [l -21. The evidence shows that this oligosaccharide, whose structure is known [3], is built up by the transfer of acetylglucosamine phosphate to dolichyl phosphate followed by the addition of one more acetylglucosamine, nine mannoses and one to three glucoses. The oligosaccharide is then transferred to protein and the glucose residues are removed. The complex oligosaccharides may then be formed by removal of six mannoses and addition of acetylglucosamine, galactose and sialic acid. Another pathway consisting in removal or addition of mannose residues would lead to the formation of the high-mannose-type oligosaccharides.This paper reports studies on some aspects of the process of glucose addition to the oligosaccharide and of transfer of the latter to protein. MATERIALS AND METHODSRat liver microsomes were prepared as described previously [4]. Dol-P was isolated and purified up to the DEAE-cellulose step [5]. D o~-P -[~~C ] G~C was prepared by incubation of UDP-[14C]Glc with liver microsomes and purified by DEAE-cellulose chromatography [5,6]. Dol-P-P-Glc-oligosaccharide labelled in the glucose or in the mannose was obtained by incubation of microsomes with UDP-['4C]G1~ [7] Abbreviations. The glucose-containing oligosaccharides with three, two and one glucose residues are referred to as Glc3, Glcz and Glcl oligosaccharides. Dol, dolichyl; Glc, glucose; Man, mannose; P, phosphate.or GDP-['4C]Man [8] respectively. Dol-P-P-Glc-oligosaccharide labelled in mannose and glucose was obtained from oviduct slices incubated with ['4C]-mannose [9,10]. Free oligosaccharides were obtained from the dolichyl diphosphate derivatives by mild acid hydrolysis [lo].Paper chromatography of the oligosaccharides was carried out with the following solvent: 1-propanol/ nitromethane/water (5 : 2 : 4). This mixture is referred to as the propanol-nitromethane solvent. RESULTSPreparation of Dolichyl-Diphosphate-Glucose-Containing Oligosaccharides. Incubation of UDP-[14C]G1~ with liver microsomes leads to the formation of labelled Dol-P-P oligosaccharides. Mild acid hydrolysis of these compounds results in the liberation of free oligosaccharides which can be separated by paper chroma...

New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus.

“…The three terminal glucose residues of the lipid-linked core oligosaccharide have been shown to be important determinants for substrate affinity to the oligosaccharyltransferase in vitro (4,(6)(7)(8)(9)(10). In vivo, the three yeast mutants accumulating Man9GlcNAc2 (aigS, alg6, and dpgl) and alg8 (accumulating Glc1Man9GlcNAc2) were found to underglycosylate secreted invertase.…”

“…Glc3Man9GlcNAc2 is the preferred substrate ofthe oligosaccharyltransferase. Removal of one, two, or all three glucose residues drastically reduces the transfer rate of the lipid-linked oligosaccharide to protein in vitro (4,(6)(7)(8)(9)(10).…”

“…The two GlcNAc and five mannose residues (derived from the nucleotide sugars UDP-GlcNAc and GDP-Man) are assembled at the cytoplasmic side, whereas the remaining four mannose residues and the three glucose residues are added in the lumen of the endoplasmic reticulum (3). These four mannose residues and the three glucose residues are transferred from dolichol phosphomannose and dolichol phosphoglucose, respectively (1,4). The addition of the three glucose residues is the last modification ofthe lipid-linked oligosaccharide before the transfer to the polypeptide.…”

Halobacterial glycoprotein biosynthesis

“…Therefore, essentially two possible functions of the transient methylation of the dolichyMinked oligosaccharides are conceivable: since dolichol derivatives do not translocate spontaneously across the lipid bilayer [41] the methylation could represent an obligatory step for translocation of the lipid oligosaccharide. Alternatively, the 3-O-methylglucose could serve a function similar to that attributed to the glucose residues on the lipid-linked Glc 3 Man 5 GlcNac 2 or GlclMan s GlcNac 2 [42,43] involved in eucaryotic glycoprotein synthesis, namely to facilitate the transfer of the oligosaccharide to protein [44][45][46][47][48][49][50][51]. It is not known whether the 3-O-methylglucose residue is removed from the oligosaccharide at the lipid-linked level or at the protein-linked level.…”

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

“…It has become evident in recent years that glycosylation of asparagine residues in eukaryotic cell proteins involves dolichol pyrophosphate (DolPP)-bound oligosaccharides as intermediates (1). It has been reported that in animal tissues (2,3), yeasts (4)(5)(6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9).…”