Protein-linked Oligosaccharides Research Articles

Defective intercellular cohesion in glycosylation mutants of Dictyostelium discoideum.

In order to identify the biological roles of protein-linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature-sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA-resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA-resistant cohesion, and reverted almost totally to wild-type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA-resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N-linked oligosaccharides in aggregation-stage intercellular cohesion.

A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues.

We have isolated and characterized a new yeast mutation in the glucosylation steps of lipid-linked oligosaccharide biosynthesis, alg8-1. Cells carrying the alg8-1 mutation accumulate Glc1Man9GlcNAc2-lipid both in vivo and in vitro. We present evidence showing that the alg8-1 mutation blocks addition of the second alpha 1,3-linked glucose. alg8-1 cells transfer Glc1Man9GlcNAc2 to protein instead of the wild type oligosaccharide, Glc3Man9GlcNAc2. Pulse-chase studies indicate that the Glc1Man9GlcNAc2 transferred is processed more slowly than the wild type oligosaccharide. The yeast mutation gls1-1 lacks glucosidase I activity (Esmon, B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327), the enzyme responsible for removing the alpha 1,2-linked glucose residues from protein-linked oligosaccharides. We demonstrate that gls1-1 cells contain glucosidase II activity (which removes alpha 1,3-linked glucose residues) and have constructed the alg8-1 gls1-1 haploid double mutant. The Glc1Man9GlcNAc2 oligosaccharide was trimmed normally in these cells, demonstrating that the alg8-1 oligosaccharide contained an alpha 1,3-linked glucose residue. A novel Glc2 compound was probably produced by the action of the biosynthetic enzyme that normally adds the alpha 1,2-linked glucose to lipid-linked Glc2Man9GlcNAc2. This enzyme may be able to slowly add alpha 1,2-linked glucose residue to protein-bound Glc1Man9GlcNAc2. The relevance of these findings to similar observations in other systems where glucose residues are added to asparagine-linked oligosaccharides and the possible significance of the reduced rate of oligosaccharide trimming in the alg mutants are discussed.

Iduronic acid: constituent of sulphated dolichyl phosphate oligosaccharides in halobacteria

The occurrence of iduronic acid in the cell surface glycoprotein of halobacteria is described. This hexuronic acid is not only found in protein-linked oligosaccharides in halobacteria but does also exist in the corresponding dolichol-linked precursors. These findings were unexpected, as iduronic acid is a typical constituent of animal glycosaminoglycans, and its biosynthesis is so far known to occur by epimerization of a glucuronic acid residue within the completed carbohydrate chain linked to protein.

Studies on N-Glycosylation by Elongating Tissues and Membranes from Pea Stems

Glucosamine and mannose were incorporated into oligosaccharides linked to either polar membrane-lipids or to asparagine residues of endogenous proteins in apical growing tissues of the etiolated pea stem. The glycolipids were subject to turnover in pulse-chase tests and protein-linked oligosaccharides accumulated with time, as expected for a precursor-product relationship. The newly formed glycoproteins were hydrolyzed by endo-beta-N-acetylglucosaminidase H to oligosaccharides in the same size range as those released by dilute acid from the lipid-linked oligosaccharides formed during the pulse. The glycoproteins were also partly degraded to free N-acetylglucosamine by beta-N-acetylhexosaminidase. Affinity of the carbohydrate moiety of the protein for concanavalin A increased between the beginning and the end of the chase, indicating processing following core glycosylation.The addition of UDP-N-acetyl-[(14)C]glucosamine plus external peptide acceptors (derived from carboxymethylated alpha-lactalbumin) to membrane preparations from the pea stem resulted in peptide glycosylation at the expense of lipid-linked oligosaccharide. Glycosylation of endogenous protein acceptors did not take place via lipid intermediates but directly from the sugar nucleotide substrate. Tunicamycin inhibited glycosyltransfer to both glycolipids and added peptides, but not to endogenous protein. It is concluded that limiting factors for N-glycosylation by pea membranes in vitro could include the unavailability of endogenous acceptors or the inability to fully elongate and internalize lipid precursors, but is not due to any limitation in capacity for N-glycosylation.

Protein-linked oligosaccharide implicated in cell-cell adhesion in two Dictyostelium species

Monoclonal antibody d-41, previously shown to block in vitro cell-cell adhesion in aggregating Dictyostelium discoideum, also blocks adhesion in aggregating D. purpureum. In both species the antibody reacts with proteins with M r ∼ 80,000, 37,000, and 27,000, presumed to be glycoproteins since the d-41 epitope is destroyed by periodate oxidation but unaffected by extensive Pronase digestion. Polyclonal antibodies raised against the mixture of d-41 reactive glycoproteins that had been purified by immunoaffinity chromatography are potent inhibitors of D. discoideum adhesion, and adhesion-blocking activity is neutralized extensively and equivalently by each of the purified glycoproteins from D. discoideum with which d-41 reacts. In contrast, polyclonal antibodies raised against the same purified glycoproteins after they had been oxidized with periodate do not block cell-cell adhesion although they react with the glycoproteins with M r ∼ 80,000, 37,000, and 27,000 and bind as extensively to the surface of aggregating D. discoideum cells as do the adhesion-blocking polyclonal antibodies. When taken together, these results raise the possibility that some component of the d-41 binding oligosaccharide participates in cell-cell adhesion.

Glycosylation of proteins in the protozoan Euglena gracilis

1.1. The main dolichol-P-P-bound oligosaccharide present in Euglena gracilis cells incubated with [U-14C]glucose was found to migrate on paper chromatography as a Glc3Man9GlcNAc2 standard and to contain glucose, mannose and N-acetylglucosamine residues.2.2. Analysis of protein-linked oligosaccharides labeled after incubating cells with [U-14C]glucose for short time periods indicated that the above-mentioned oligosaccharide was transferred to protein.3.3. The mechanism of protein N-glycosylation in this protozoan is described. It is similar to that known to occur in higher eukaryotic cells and different from that described for other protozoa.

Yeast mutants deficient in protein glycosylation.

The synthesis of asparagine-linked oligosaccharides involves the formation of a lipid-linked precursor oligosaccharide that has the composition Glc3Man9GlcNAc2. We have used a [3H]mannose suicide selection to obtain mutants in yeast that are blocked in the synthesis of this precursor oligosaccharide. The alg1 mutant accumulated lipid-linked GlcNAc2, alg2 mutants accumulated Man1-2GlcNAc2, alg3 mutants accumulated Man5GlcNAc2, alg4 mutants accumulated Man1-8GlcNAc2, and alg5 and alg6 mutants accumulated Man9GlcNAc2. Some of these mutants appeared to transfer oligosaccharides other than Glc3Man9GlcNAc2 from the lipid carrier to invertase. These aberrant protein-linked oligosaccharides were processed by the addition of outer chain residues in the alg3, alg5, and alg6 mutants. There was virtually no outer chain addition in the alg2 and alg4 mutants. alg4 was the only mutant that failed to secrete invertase.

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly.

The formation of N-glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

Regulation of glycoprotein biosynthesis by formation of specific glycosyltransferase complexes.

Eukaryotic surfaces simultaneously express a complex array of protein-linked oligosaccharides and, during differentiation and on neoplastic transformation, these arrays change. The oligosaccharides linked to asparagine residues of proteins are a complex family of structures that are derived from a common precursor oligosaccharide by a network of competing biosynthetic pathways. The glycosylation reactions that make up these biosynthetic pathways often share common intermediates, requiring the competition between rival glycosyltransferases to be regulated at the supramolecular level. Investigation of two sequential glycosyltransferases that together add N-acetyllactosamine to glycoproteins has revealed their ability to form specific complexes. The functional consequences of complex formation were assessed by investigating the coupled reaction carried out by these sequential enzymes. The membrane enzymes are readily adsorbed by preformed liposomes, and their ability to interact after liposome adsorption has allowed direct investigation of the action carried out by these sequential enzymes when they are coadsorbed to the same liposome or adsorbed to separate liposomes shows the preferential use of endogenously generated intermediate over exogenously added glycoproteins. This facilitated passage of the intermediate glycosylated glycoprotein within the complex results in a tight coupling of the sequential enzymes. The formation of such complexes by sequential glycosyltransferases is proposed as a mechanism for controlling the competition between potentially rival glycosylation sequences during glycoprotein synthesis. The formation of different specific complexes under different conditions provides a flexible mechanism for regulating the synthesis of cell surface glycoproteins during development.

Chapter 7 Synthesis and Processing of Asparagine-Linked Oligosaccharides of Glycoproteins

Publisher Summary This chapter discusses three stages in the synthesis of asparagine-linked carbohydrates: (1) synthesis of the large lipid-linked oligosaccharide, (2) glycosylation of asparagine residues by the transfer of oligosaccharide from lipid to protein, and (3) processing of protein-linked oligosaccharides to yield mature high-mannose and complex glycopeptides. The asparagine-linked oligosaccharides of cell surface and extracellular glycoproteins are a heterogeneous class of molecules that can be divided in two basic types. High-mannose structures have a Man 2 GlcNAc 2 inner core at the reducing end and two to six additional mannose residues in a peripheral three-branched structure. Complex oligosaccharides also have a Man 3 GlcNAc 2 inner core and in addition contain a fucose residue and two to four terminal trisaccharide branches containing N -acetylglucosamine, galactose, and sialic acid. Both types of oligosaccharides show microheterogeneity; a number of related structures are usually found in a single purified glycoprotein preparation. The number of mannose residues in high-mannose oligosaccharides is variable, whereas complex oligosaccharides show variability in sialic acid and fucose content.

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.

Glycoprotein biosynthesis. Rat liver microsomal glucosidases which process oligosaccharides.

Asparagine-linked oligosaccharides of glycoproteins undergo extensive modification or "processing" following their attachment to protein. A key step in post-glycosylation processing is the sequential removal of glucose residues from the protein-linked oligosaccharide. We have studied rat liver preparations which catalyze removal of glucose from Glc3Man9GlcNAc, Glc2Man9GlcNAc, and Glc1Man9GlcNAc. Detergent solubilization studies, inhibitor studies, and temperature-activity profiles indicate that at least two distinct glucosidases are present in the membranes. One of these glucosidases removes the distal glucose from Glc3Man9GlcNAc, and the other glucosidase sequentially removes glucose from Glc2Man9GlcNAc and Glc1Man9GlcNAc. The latter glucosidase has been solubilized from the microsomal memrbranes and purified 12-fold. The glucosidases, which are integral membrane proteins, are localized in the rough and smooth microsomes and appear to be located on the cisternal surface of the microsomal vesicles. These glucosidases are suggested to be of biological importance in catalyzing the initial events in the post-glycosylation processing of cellular glycoprotein.

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: I. Mannosylated units

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[ 14C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [ 14C]Man material specifically soluble in CHCl 3/CH 3OH/H 2O, 10 : 10 : 3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some dolichol-P-[ 14 C] Man ). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to α-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N, N′-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [ 14C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: II. Glucosylated units

Pig thyroid rough microsomes catalyzed the transfer of glucose from UDP-[ 14C]Glc to glycolipids extractable with chloroform/methanol, glycolipids extractable with a water-saturated chloroform/methanol and to a residual material. Kinetics of labeling were compatible with a precursor-product relationship between the second type of glycolipid and residuals. The [ 14C] Glc-glycolipids soluble in CHCl 3/CH 3OH/H 2O, 10 : 10 : 3, behaved on DEAE-cellulose mainly as pyrophospho derivatives, with some less acidic radioactivity, probably dolichol-P-[ 14 C] Glc . Their saccharide moieties released by mild acid appeared polydisperse on paper chromatography, a part of them being estimated larger than a nonasaccharide marker GlcNAc-[Man] 8. The 14C-labeled glucosylated glycoproteins have represented all the considerable polymeric label remaining after lipid extraction. Their pronase glycopeptides were submitted to a differential reductive alkaline hydrolysis and it was concluded that their [ 14C] glucose belongs mainly to N-glycosically linked units. On gel filtration, the released saccharides exhibited an average size of nine monosaccharide units (from six to twelve with a relatively high proportion of material containing more than nine sugars). In a [ 14C] Glc-microsomal extract, 29% of the non-lipid radioactivity was found immunoreactive with an antiserum to pig thyroglobulin.

The formation of oligoglucans linked to lipid during synthesis of β-glucan by characterized membrane fractions isolated from peas

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble beta-glucans, which like the oligosaccharides contained beta(1leads to 3) and beta-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in beta-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.