N-Glycosylation of membrane glycoproteins in retinol-deficient rat liver

The effect of vitamin A deficiency onN-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in proteinN-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled withd-[2-³H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc₁Man₉GlcNAc₂, Man₉GlcNAc₂, Man₈GlcNAc₂ and Man₇GlcNAc₂, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number ofN-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis ofN-linked oligosaccharides of glycoproteins must be questioned.Abbreviations DolP Dolichyl phosphate - DolPP dolichyl pyrophosphoryl - RetPMan retinyl phosphate mannose - DPP IV dipeptidyl peptidase IV (EC 3.4.14.5) - endo H endo--N-acetylglucosaminidase H (EC 3.2.1.96) - endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Degradation of Misfolded Endoplasmic Reticulum Glycoproteins in Saccharomyces cerevisiae Is Determined by a Specific Oligosaccharide Structure

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man₈GlcNAc₂ oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man₆GlcNAc₂, Man₇GlcNAc₂ and Man₉GlcNAc₂ oligosaccharides, whereas Man₈GlcNAc₂ and, to a lesser extent, Man₅GlcNAc₂ oligosaccharides supported degradation. These results suggest a role for the Man₈GlcNAc₂ oligosaccharide in the degradation process. They may indicate the presence of a Man₈GlcNAc₂-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.     

Biosynthesis and characterization of large dolichyl diphosphate-linked oligosaccharides in Sacchromyces cerevisiae

Yeast membranes incorporate radioactivity from GDP[¹⁴C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components.The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditios. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol.When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharide is formed besides the previously-described Dol-PP-(GlcNAc₂ mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [¹⁴C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.     

NovelN-linked oligo-mannose type oligosaccharides containing an α-d-galactofuranosyl linkage found in α-d-galactosidase fromAspergillus niger

Structures of oligosaccharides fromAspergillus niger -d-galactosidase [EC 3.2.1.22] were studied. Purified -d-galactosidase was treated withN-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by¹H-NMR and compositional analysis to be Man₅GlcNAc₂, Man₆GlcNAc₂, Man₉GlcNAc₂, GlcMan₉GlcNAc₂, GalMan₄GlcNAc₂ and GalMan₅GlcNAc₂. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an 1–2 linkage (GalfMan₄GlcNAc₂ and GalfMan₅GlcNAc₂).     

Biosynthesis of mannose-containing lipid-linked oligosaccharides by solubilized enzyme preparation from cultured soybean cells

Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at −10°C for 1 month. The solubilized enzyme synthesized [¹⁴C]Man₃GlcNAc₂-pyrophosphoryl-polyprenol and [¹⁴C]Man₅GlcNAc₂-pyrophosphoryl-polyprenol when incubated with GDP-[¹⁴C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man₃GlcNAc₂-lipid and Man₅GlcNAc₂-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man₆GlcNAc₂-lipid, Man₇GlcNAc₂-lipid, and Man₈GlcNAc₂-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man₅GlcNAc₂-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.     

Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. AfterN-acetylation, the oligosaccharides were labelled with a UV-absorbing compound,p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz¹H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc1-3Man_7–9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man_5–9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man1-6(±GlcNAc1-4)(Man1-3)Man1-4GlcNAc1-4(±Fuc1-6)GlcNAc. The glucosylated oligosaccharides, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂, have not previously been reported in mature glycoproteins from any source.Abbreviations IgG, IgM, IgD, IgE, and IgA immunoglobulin G, M, D, E, and A, respectively - IgY egg-yolk antibody - ABEE p-aminobenzoic acid ethyl ester - HPLC high performance liquid chromatography - FAB-MS fast atom bombardment mass spectrometry - Hex hexose - HexNAc N-acetylhexosamine - hCG human chorionic gonadotropsin     

Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas.

Studies reported in the preceding paper (Trowbridge and Hyman, 1979) have demonstrated that Thy-1^? mutant lymphoma cells of the class E complementation group lack the normal high molecular weight lipid-linked oligosaccharide, but instead accumulate two smaller species termed I and II. This paper reports studies which elucidate the structures of lipid-linked oligosaccharides I and II. By subjecting oligosaccharides radiolabeled with ³H-mannose, ³H-glucose or ³H-glucosamine to methylation, acetolysis, periodate oxidation and exoglycosidase digestion, the structures were shown to be: where R = GlcNac B1,4(3) GlcNAc. A comparison of I and II with lipid-linked oligosaccharides from normal Chinese hamster ovary cells indicates that both I and II are normal biosynthetic intermediates. On the basis of these data we suggest that the defect in the class E mutant cells is the lack of an α1,3 mannosyltransferase involved in the conversion of the Man₅GlcNAc₂ lipid-linked oligosaccharide to the Man₆GlcNAc₂ intermediate. It is also impossible that the same enzyme is involved in conversion of the Glc₃Man₅GlcNAc₂ lipid-linked oligosaccharide to Glc₃Man₆GlcNAc₂. The latter reaction, however, has not yet been demonstrated in normal cells.     

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc₃Man₉GlcNAc₂ precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-³H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man_9-8GlcNAc₂. In contrast, unstable ERAD substrates are trimmed to Man_6-5GlcNAc₂ and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.Download video file.^{(118M, mp4)}     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide

• 1.1. A maximum rate of dolichyl phosphate [¹⁴C]glucose synthesis from 55-day embryos was achieved at 16nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate.
• 2.2. The highest values of [¹⁴C]glucose incorporation from UDP-[¹⁴C]glucose into dolichyl phosphate [¹⁴C]glucose, dolichyl diphosphate [¹⁴C]Glc-oligosaccharides and proteins were reached at 5 min time point of incubation of liver microsomes both from embryos and sows.
• 3.3. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows.
• 4.4. The enzymatic transfer of Glc₃-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides.
• 5.5. One labelled compound was discovered in the Chcl₃-Ch₃Oh-H₂O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[¹⁴C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc₂Man₉Glc₃.

     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Processing of asparagine-linked saccharides in Mucor rouxii

Asparagine-linked Glc₁Man₉GlcNAc₂, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂ were detected in mycelial-form cells of the dimorphic fungus Mucor rouxii inculbated with [U-¹⁴C]glucose for 3 min. The oligosaccharides were absent from glycoproteins isolated from cells chased for 15 min with the unlabed monosaccharide. This was due to deglucosylation of the oligosaccharides and not to further addition of mannose residues to them. The half-lives of the glucosylated compounds were much shorter, therefore, in M. rouxii than in other eucaryotic cells. Further processing of N-linked saccharides led to the synthesis of mannan-like glycoproteins, some of whch contained methyl groups in position 3 or the mannose residues. Methylation occurred only at the non-reducing ends and prevented further elongation of the branches.     

The purification of the three trimannosyl-N-acetylchitobiose pentasaccharides from the urine of swainsonine-intoxicated sheep

A simple method for the preparative resolution of three Man₃GlcNAc₂ isomers called Ia, Ib and II has been designed. It consists mainly of the use of concanavalin A-Sepharose which allowed the total purification of Man₃GlcNAc₂-Ia, and then of anion-exchange resin in borate buffer-gradient to separate the Ib and II isomers. The purity of each oligosaccharide was checked by two HPLC methods. The use of these oligosaccharides for different analytical and biosynthetic purposes is discussed, and the unexpected resistance of one of the Man₃GlcNAc₂ alditols to the action of endo--N-acetylglucosaminidase H is noted.     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate.

Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group.     

Overexpression of a homogeneous oligosaccharide with <Superscript>13</Superscript>C labeling by genetically engineered yeast strain

This report describes a novel method for overexpression of ¹³C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly ¹³C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man₈GlcNAc₂ oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, ¹³C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific ¹³C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The ¹³C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.     

Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional ��1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc₃Man₉ GlcNAc₂-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the α1,3- and the α1,6-mannose-linked residue from GDP-mannose to Man₁GlcNAc₂-PP-Dol forming the Man₃GlcNAc₂-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX₇E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys²³⁰, as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1–4), and several features of this pathway even occur in prokaryotes (5–7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc₃Man₉ GlcNAc₂-PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8–10). The initial steps of the dolichol pathway up to Man₅GlcNAc₂-PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER),² using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14–17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18–20).One of the temperature-sensitive alg mutants, alg2, was shown to accumulate lipid-linked Man₂GlcNAc₂ at the restrictive temperature (15), indicating that alg2 might have a defect in the glycosyltransferase catalyzing the transfer of the third, α1,6-linked mannose, i.e. in the formation of the branched pentasaccharide Man₃GlcNAc₂-PP-Dol (see Fig. 8). On the other hand, biochemical studies in human fibroblasts from a patient with a defect in the human ALG2 ortholog, causing congenital disorder of glycosylation type CDG1i, pointed to a role in the transfer of the second, α1,3-linked mannose residue, because no elongation of Man(1,6)ManGlcNAc₂-PP-Dol occurred (21). In contrast, control fibroblasts were able to do so, albeit with reduced efficiency when compared with Man(1,3)ManGlcNAc₂-PP-Dol as glycosyl acceptor. Because a bioinformatic approach of the yeast data base did not reveal an unknown open reading frame that might encode an additional putative mannosyltransferase being involved in LLO synthesis, we reasoned that ALG2 may have a dual function, i.e. synthesizing both Man₂GlcNAc₂-PP-Dol and Man₃GlcNAc₂-PP-Dol. While the current study was in progress, evidence was presented that a membrane fraction from Escherichia coli, expressing ALG2 from yeast, is able to carry out an α1,3- and α1,6-mannosylation to form the branched pentasaccharide intermediate (22). However, the contribution of native E. coli enzymes could not entirely be ruled out. So far Alg2 has not been studied biochemically in yeast. Here, we confirm and extent this finding by investigating Alg2 in yeast. We first established a radioactive in vitro assay and demonstrate that Alg2, immunoprecipitated from detergent extracts of yeast microsomal membranes, is indeed sufficient to catalyze both elongation of Man₁GlcNAc₂-PP-Dol to Man₂GlcNAc₂-PP-Dol and subsequently to Man₃GlcNAc₂-PP-Dol. Furthermore we investigated the membrane topology of Alg2 mannosyltransferase. Evidence will be presented that Alg2 is composed only of the two N-terminal of four predicted transmembrane domains (TMDs), whereas the C-terminal hydrophobic sequences contribute to ER localization merely in a nontransmembrane manner. Surprisingly, none of the four domains is essential for Alg2 activity because deletion of either the two N-terminal or C-terminal domains gives rise to an active transferase. Finally, we perform a mutational analysis of Alg2 and identify amino acids required for its activity.Open in a separate windowFIGURE 8.Early steps of lipid-linked oligosaccharide formation on the cytosolic side of the ER membrane. Biosynthesis starts with the transfer of a GlcNAc-phosphate to dolichol phosphate with formation of the pyrophosphate bond, catalyzed by Alg7. The second step is catalyzed be the dimeric Alg14/Alg13 complex, whereby membrane-bound Alg14 recruits cytosolic Alg13 to the membrane with formation of the active GlcNAc transferase. Following the addition of the β1,4-linked mannose by Alg1, Alg2 catalyzes, as demonstrated here, both the transfer of the α1,3- and α1,6-linked mannose. The two final α1,2-mannose residues are transferred by Alg11, before the Man₅GlcNAc₂-PP heptasaccharide is translocated across the ER membrane to the lumen, where further elongation takes place to the full-length core saccharide. All of the sugar residues are donated by sugar nucleotides.     

Structure of carbohydrate chains of fucolectin from the bark of golden rain shrub <Emphasis Type="Italic">Laburnum anagyroides</Emphasis>

A reductive LiBH₄-Bu^tOH cleavage of N-glycosylamide carbohydrate-peptide bond allowed splitting off of oligosaccharide chains of the fucolectin, the bark agglutinin from the shrub golden rain Laburnum anagyroides (LABA). Four N-glycans were isolated by HPLC, and their structures were elucidated by monosaccharide analysis and ¹H NMR (500 MHz) spectroscopy: Man₂Fuc₁Xyl₁GlcNAc₂ (M2FX), Man₃Xyl₁GlcNAc₂ (M3X), Man₃Fuc₁Xyl₁GlcNAc₂ (M3FX), and Man₃Xyl₁Fuc₁GlcNAc₃ (NM3FX). All the N-glycans contain D-xylose and three of them, L-fucose; they were found to be in a 1:8:3:1 ratio.