Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Lehle, Ludwig; Schulz, I.; Tanner, Widmar

doi:10.1007/bf00427198

Cited by 20 publications

(7 citation statements)

References 43 publications

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“…The proposed scheme is supported by studies in vivo showing synthesis of identical lipid-linked oligosaccharides after labelling cells with mannose or glucose [48].…”

Section: Discussionmentioning

confidence: 74%

Biosynthesis of the Core Region of Yeast Mannoproteins. Formation of a Glucosylated Dolichol-Bound Oligosaccharide Precursor, Its Transfer to Protein and Subsequent Modification

Lehle

1980

Eur J Biochem

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117

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A new membrane preparation from Saccharomyces cerevisiae was developed, which effectively catalyzes the synthesis of large oligosaccharide-lipids from GDP-Man and UDP-Glc allowing a detailed study of their formation and size.The oligosaccharide from an incubation with GDP-Man could be separated by gel filtration chromatography into several species consisting of two N-acetylglucosamine (GlcNAc) residues at the reducing end and differing by one mannose unit; the major compound formed has the composition (Man)g(GlcNAc)2. Upon incubation with UDP-Glc, three oligosaccharides corresponding to the size of (G1c)l -s(Man)9(GlcNAc)2 are formed. Thus, the oligosaccharides generated in vitro by the yeast membranes appear to be identical in size with the oligosaccharides found in animal systems. In addition the results indicate that dolichyl phosphate mannose (DolP-Man) is the immediate donor in assembling the oligosaccharide moiety from (Man)5(GlcNAc)2 to (Man)9(GlcNAc)2. All three glucose residues are transferred from DolP-Glc. Experiments with isolated [Gl~-~~C]oIigosaccharide-lipid as substrate demonstrated that theoligosaccharide chain is transferred to an endogenous membrane protein acceptor. Moreover, transfer is followed by an enzymic removal of glucose residues, due to a glucosidase activity associated with the membranes. Glucose release from the free [Glc-14C]oligosaccharide is less effective than from protein-bound oligosaccharide. Glycosylation was also observed using the [Man-14C]oligosaccharide-lipid or D O~P P -( G I~N A C )~ as donor. However, transfer in the presence of glucose seems to be more rapid.The mannose-containing oligosaccharide, released from the lipid, was shown to function as a substrate for further chain elongation reactions utilizing GDP-Man but not DolP-Man as donor.It is suggested that the immediate precursor in the synthesis of the heterogeneous core region, (Man)12 -17(GlcNAc)2, of yeast mannoproteins is a glucose-containing lipid-oligosaccharide with the composition (Gl~)3(Man)9(GlcNAc)~, i.e. only part of what has been defined as inner core is built up on the lipid carrier. After transfer to protein the oligosaccharide is modified by excision of the glucose residues, followed subsequently by further elongation from GDP-Man to give the size of the oligosaccharide chains found in native mannoproteins.Studies from several laboratories have recently shown that asparagine-linked carbohydrate chains of cellular and extracellular glycoproteins seem to be derived from a common precursor, a glucose-containing lipid-linked oligosaccharide [l -71 with the comAhhreviurions. DolP, dolichyl monophosphate; DolPP, dolichyl diphosphate; Man, mannose; Glc, glucose; GlcNAc, N-acetylglucosamine; (GlcNAc)z, chitobiose; GDP-Man, guanosine diphosphate mannose; UDP-Glc, uridine diphosphate glucose; Glcoligosaccharides, (Glc), -3 (Man)q(GlcNAc)z ; Man-oligosaccharides, (Man)., (GlcNac)>; VSV, vesicular stomatitis virus. position (Gl~)3(Man)~(GlcNAc)~ [8,9]. After the transfer en bloc from the lipid (dolichol) ca...

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“…The proposed scheme is supported by studies in vivo showing synthesis of identical lipid-linked oligosaccharides after labelling cells with mannose or glucose [48].…”

Section: Discussionmentioning

confidence: 74%

Biosynthesis of the Core Region of Yeast Mannoproteins. Formation of a Glucosylated Dolichol-Bound Oligosaccharide Precursor, Its Transfer to Protein and Subsequent Modification

Lehle

1980

Eur J Biochem

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117

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“…Andererseits werden unter den von uns in der vorliegenden Arbeit gewahlten alkalischen Abbaubedingungen nicht nur die iiber Ser/Thr 0-glycosidisch und uber Phosphodiester gebundenen Saccharide abgespalten, sondern auch Oligosaccharide, die N-glycosidisch uber die Amidgruppe des Asparagins mit der Peptidkette verknupft sind. Auf Grund der erhaltenen Ergebnisse erweitern wir das fur Mannan aus Naccharomyces cerevisiae (LEHLE et al 1980) erhaltene Biosynthesemodell besonders bezuglich der Bedeutung von Man-6P als Aktivator und Mannosyl-( oder Mannosyl-P-) Donator fur die mikrosomale Synthese der alkalilabil gebundenen Oligosaccharide und des hochpolymeren alkalistabilen Mannans.…”

Section: B R~r E R Und G Reuterunclassified

Effektor‐ und Precursorfunktion von Mannose‐6‐phosphat, Mannose‐l‐phosphat, UDP‐N‐acetylglucosamin und Dolichylphosphat bei der mikrosomalen Biosynthese von Proteophosphomannan der SCP‐Hefe Candida maltosa H

Röber

Reuter

1985

J. Basic Microbiol.

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Microsomal preparations from protoplasts of the yeast Candidn maltosu, which is able to assimilate n-alkanes, were used as enayme source to catalyse the incorporation of radioactivity from guanosine diphosphato "%-mannose, ~HH-mannose-6-phosphate, SH-mannose-l-phosphate or uridine diphosphate N-ttcctyl-( 14C-glucose)-glusosaminc in a mannose polysaccharide. The reaction product was identified as yeast mannan by comparing the saccharides prepared by alkaline degradation and by acetolysis, with those from in vivo proteophosphomannan, obtained in structural investigations.The enzyme preparation catalysed the formation of an almost unsubstituted LZ( 1,6)-linked mannan backbone from GDP-Man. Man-GP, dolichylphosphate and on the other hand UDP-CNAc in the presence of ATP and dolichylphosphate are potent activators of the mannosyl transfer: n(1,t)- (and a(1,3)-) mannosyl transferases are activated by Man-6P. Beside this Man-6P is found to be a precursor for the incorporationof the first mannosyl (-phosphate) unit in the side chain of a branched mannan and of the alkali-labile bound oligosaccharides.The data presented support the hypothesis that Man-GP, not activated by GTP, is a second munnosyldonator in the biosynthesis of proteophosphomannan.

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“…Firstly, why are dolichyl phosphates as 'lipid intermediates' involved in this biosynthetic process? Secondly, what is the crucial general importance of protein glycosylation, evident from the fact that the dolichol pathway has obviously been preserved in a very conservative way during evolution (Lehle et al 1980) ? Experiments related to both these questions will mainly be discussed in this paper.…”

Section: Introductionmentioning

confidence: 99%

“…The existence of dolichyl-PP-oligosaccharides containing GlcNAc, M an and Glc have been described (Parodi 1977(Parodi , 1981Lehle 1980Lehle , 1981 and the oligosaccharides synthesized in vitro and in vivo have been shown to be of the same size as the mammalian GlcNAc2M an9Gl (Lehle et al 1980).…”

Section: Introductionmentioning

confidence: 99%

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins

Tanner

Haselbeck

Schwaiger

et al. 1982

Phil. Trans. R. Soc. Lond. B

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The pathways for protein N- and O-glycosylation in yeast cells are summarized. Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated. A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues. The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously. However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction. The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle. In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth. This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase. It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.

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Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Cited by 20 publications

References 43 publications

Biosynthesis of the Core Region of Yeast Mannoproteins. Formation of a Glucosylated Dolichol-Bound Oligosaccharide Precursor, Its Transfer to Protein and Subsequent Modification

Biosynthesis of the Core Region of Yeast Mannoproteins. Formation of a Glucosylated Dolichol-Bound Oligosaccharide Precursor, Its Transfer to Protein and Subsequent Modification

Effektor‐ und Precursorfunktion von Mannose‐6‐phosphat, Mannose‐l‐phosphat, UDP‐N‐acetylglucosamin und Dolichylphosphat bei der mikrosomalen Biosynthese von Proteophosphomannan der SCP‐Hefe Candida maltosa H

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins

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