Formation of Lipid-Bound Oligosaccharides Containing Mannose. Their Role in Glycoprotein Synthesis

Behrens, Nicolas; Carminatti, Héctor; Staneloni, Roberto J.; Leloir, Luis F.; Cantarella, Ana I.

doi:10.1073/pnas.70.12.3390

Cited by 145 publications

(68 citation statements)

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“…The oligosaccharides obtained by mild acid hydrolysis were separated by paper chromatography with the solvent I-butanol/pyridine/water (4:3 :4) for 20 days oligosaccharide to protein and found that its size corresponded to the Gk3-containing compound. Transfer of mannose-labelled oligosaccharides presumably not containing glucose has been reported [8,19]. Their rate of transfer is slower and may involve endogenous glucose donors.…”

Section: Discussionmentioning

confidence: 99%

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Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein

Staneloni

Ugalde

Leloir

1980

European Journal of Biochemistry

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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...

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Section: Discussionmentioning

confidence: 99%

“…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].…”

Section: Methodsmentioning

confidence: 99%

Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein

Staneloni

Ugalde

Leloir

1980

European Journal of Biochemistry

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“…They were revealed as described in the legend to Fig. 2 previously performed with the dolichol-PP derivatives from rat liver containing mannose [12] or with the similar compounds from rat liver or yeast containing in addition glucose residues [26,27]. In the case of the latter liver compound, gas-liquid chromatography performed on large quantities of the compound confirmed the presence of two N-acetylglucosamine residues [28].…”

Section: Presence Of N-acetylhexosamine Residuesmentioning

confidence: 97%

“…The acceptors added corresponded to those contained in I5 g of rat liver. The reaction products containing two phosphates were separated from the monophosphate derivatives by chromatography in 45 x 1.2 cm DEAE-cellulose (acetate form) column as described by Behrens et al [12].…”

Section: Large Scale Preparationmentioning

confidence: 99%

Lipid Intermediates in the Synthesis of the Inner Core of Yeast Mannan

Parodi

1978

Eur J Biochem

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The synthesis by yeast microsomes of compounds that are probably dolichol-pyrophosphate derivatives containing N,N '-diacetylchitobiose and several mannose residues is described. However, the presence of monosaccharide residues other than N-acetylglucosamine and mannose has not been ruled out. The amount of the lipid derivatives synthesized was enhanced by the addition to the incubation mixture of an organic-solvent-soluble extract from rat liver known to contain dolicholpyrophosphate oligosaccharides. Incubation of these derivatives with yeast microsomes led to the transfer of about fifteen percent of their saccharide moieties to endogenous proteins. The oligosaccharides released from the dolichol derivatives by mild acid hydrolysis could serve as primers for the synthesis of a polysaccharide having the characteristics of mannan outer-chain. It is suggested that the dolichol-pyrophosphate derivatives described in this paper are intermediates in the synthesis of mannan inner core.

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“…Rat liver microsomes have been shown to synthesize lipid-linked pyrophosphate diesters of GlcNAc and di-Nacetylchitobiose (6). Lipid-bound oligosaccharide phosphates have also been isolated (3,7,8,9). Similar compounds found in bacterial and plant systems are involved in the formation of other complex glycans (10).…”

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Enzymatic Utilization of P ¹ -Di- N -acetylchitobiosyl P ² -Dolichyl Pyrophosphate and Its Chemical Synthesis

Wedgwood

Warren

Jeanloz

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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Fully acetylated chitobiose was treated with phosphoric acid to give a mixture of products from which 2-acetamido-4-0-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-fB-D-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy -a,#-D-glucopyranosyl phosphate (Ac3GlcNAc-Ac2GlcNAc-P) was isolated by preparative thin layer chromatography.Treatment of this compound with P1-diphenyl P2-dolichyl pyrophosphate gave an acetylated pyrophosphate diester, which was purified chromatographically and deacetylated.The product, P'-di-N-acetylchitobiosyl P2-dolichyl pyrophosphate (Dol-P-P-GlcNAc-GlcNAc), was readily separated from P'-2-acetamido-2-deoxy-a-D.glucopyranosyI P2-dolichyl pyrophosphate (Dol-P-P-GlcNAc) by thin layer chromatography in several solvent systems. Addition of this compound to human lymphocyte membrane preparations led to the formation of a mannose-containing derivative which appears to be an oligosaccharide phospholipid, as judged by its behavior on DEAE-cellulose chromatography and by its hydrolysis to give an oligosaccharide containing more than four monosaccharide units.Incubations of membrane preparations from a variety of animal sources with nucleoside diphosphate sugar compounds give glycosyl polyprenyl phosphates, which are intermediates in the biosynthesis of some glycoproteins (1-3). Dolichyl f-Dmannopyranosyl phosphate (Dol-P-Man) has been identified as a product of the incubation with GDP-Man in calf pancreas and in human lymphocytes (4). Dol-P-P-GlcNAc has been shown to be formed from UDP-GlcNAc in calf pancreas (5). Rat liver microsomes have been shown to synthesize lipid-linked pyrophosphate diesters of GlcNAc and di-Nacetylchitobiose (6). Lipid-bound oligosaccharide phosphates have also been isolated (3,7,8,9). Similar compounds found in bacterial and plant systems are involved in the formation of other complex glycans (10).It has been suggested that a mannose-containing oligosaccharide lipid is formed by the transfer of mannose from Abbreviations: GlcNAc-P, 2-acetamido-2-deoxy-D-glucopyranosyl phosphate; Ac3GlcNAc-P, 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl phosphate; Ac3GlcNAc-Ac2GlcNAc-P and GlcNAc-GlcNAc-P, peracetyl-and di-N-acetyl-a,,2-chitobiosyl phosphates, respectively; Dol-P, dolichyl phosphate; Dol-P-Man, dolichyl f3-D-mannopyranosyl phosphate; Dol-P-P, dolichyl pyrophosphate; Dol-P-P-GlcNAc, PL-2-acetamido-2-deoxy-a-D-glucopyranosyl P2-dolichyl pyrophosphate; Dol-P-P-GlcNAc-GlcNAc, PL-di-N-acetylchitobiosyl P2-dolichyl pyrophosphate [P'-2-acetamido-4-0-(2-acetamido-2-deoxy-j3-Dglucopyranosyl)-2-deoxy-a,j2-D-glucopyranosyl P2-dolichyl pyro GDP-Man or Dol-P-Man to Dol-P-P-GlcNAc-GlcNAc (8). An authentic sample of Dol-P-P-GlcNAc-GlcNAc has been obtained by chemical synthesis. This compound stimulates the incorporation, in human lymphocyte membrane preparations, of mannose into a compound with the properties of an oligosaccharide phospholipid containing more than four monosaccharide units. A similar oligosaccharide phospholipid, which contains GlcNAc as well as mannose, is formed when...

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Formation of Lipid-Bound Oligosaccharides Containing Mannose. Their Role in Glycoprotein Synthesis

Cited by 145 publications

References 16 publications

Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein

Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein

Lipid Intermediates in the Synthesis of the Inner Core of Yeast Mannan

Enzymatic Utilization of P ¹ -Di- N -acetylchitobiosyl P ² -Dolichyl Pyrophosphate and Its Chemical Synthesis

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Formation of Lipid-Bound Oligosaccharides Containing Mannose. Their Role in Glycoprotein Synthesis

Cited by 145 publications

References 16 publications

Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein

Addition of Glucose to Dolichyl Diphosphate Oligosaccharide and Transfer to Protein

Lipid Intermediates in the Synthesis of the Inner Core of Yeast Mannan

Enzymatic Utilization of P 1 -Di- N -acetylchitobiosyl P 2 -Dolichyl Pyrophosphate and Its Chemical Synthesis

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Enzymatic Utilization of P ¹ -Di- N -acetylchitobiosyl P ² -Dolichyl Pyrophosphate and Its Chemical Synthesis