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PubMed2003

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors.

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta 4-glucuronic acid-beta 3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10-20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (approximately 1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.

Bioreactors↗
PubMed2006

Role of the carbohydrate binding site of the Streptococcus pneumoniae capsular polysaccharide type 3 synthase in the transition from oligosaccharide to polysaccharide synthesis.

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.

Bacterial Capsules↗
PubMed2006

Heparan sulfate-related oligosaccharides in ternary complex formation with fibroblast growth factors 1 and 2 and their receptors.

Biosynthesis of heparan sulfate (HS) is strictly regulated to yield products with cell/tissue-specific composition. Interactions between HS and a variety of proteins, including growth factors and morphogens, are essential for embryonic development and for homeostasis in the adult. Fibroblast growth factors (FGFs) and their various receptors (FRs) form ternary complexes with HS, as required for receptor signaling. Libraries of HS-related, radiolabeled oligosaccharides were generated by chemo-enzymatic modification of heparin and tested for affinity to immobilized FR ectodomains in the presence of FGF1 or FGF2. Experiments were designed to enable assessment of N-sulfated 8- and 10-mers with defined numbers of iduronic acid 2-O-sulfate and glucosamine 6-O-sulfate groups. FGF1 and FGF2 were found to require similar oligosaccharides in complex formation with FR1c-3c, FGF2 affording somewhat more efficient oligosaccharide recruitment than FGF1. FR4, contrary to FR1c-3c, bound oligosaccharides at physiological ionic conditions even in the absence of FGFs, and this interaction was further promoted by FGF1 but not by FGF2. In all systems studied, the stability of FGF-oligosaccharide-FR complexes correlated with the overall level of saccharide O-sulfation rather than on the precise distribution of sulfate groups.

Animals↗
PubMed1996

Fate of fructo-oligosaccharides in the human intestine.

There is a need for studies on colonic fermentation in order to learn more about health and diseases of the colon. The aim of the present study was to evaluate the fate of two different doses of fructo-oligosaccharides (5 and 15 g/d) v. glucose in the intestine of healthy men. Twenty-four volunteers participated in a 5-week study. The study was a completely balanced multiple crossover trial using an orthogonal Latin-square design for three periods, with supplement periods of 7 d and two 7 d wash-out periods. Breath samples and faecal samples were collected. There was a clear gaseous response to the consumption of fructo-oligosaccharides. The highest dose significantly increased 24 h integrated excretion of breath H2 (P < 0.05). Breath H2 excretion after ingestion of 5 g fructo-oligosaccharides was higher than control, but did not reach significance. No effects on the total concentration of short-chain fatty acids in faeces were observed, no modification of the molar proportions of the various short-chain fatty acids was observed. The faecal pH did not change. No changes in faecal weight were observed. No fructo-oligosaccharides were recovered in faeces. We conclude that fructo-oligosaccharides added to the diet of young Western subjects are fully metabolized in the large intestine. The level of fermentation seem to be dose-dependent.

Adult↗
PubMed1997

Compared effects of three oligosaccharides on metabolism of intestinal microflora in rats inoculated with a human faecal flora.

Using germ-free rats inoculated with a human faecal flora (gnotobiotic rats), the effects of three oligosaccharides (beta-fructo-oligosaccharides (FOS), beta-galacto-oligosaccharides (TOS) and alpha-gluco-oligosaccharides (GOS)) on intestinal bacterial metabolism were compared. The animals were fed on either a control diet or diets containing 40 g/kg of GOS, FOS or TOS. FOS and TOS were the preferred growth substrates for Bifidobacteria which increased in number by 2 log values in faeces of rats when compared with rats fed on GOS or control diets. Ingestion of TOS specifically induced hydrolysis of the substrate, and did not modify the activity of any other enzymes measured in the caecum. GOS led to a non-specific enzymic induction of beta-galactosidase (EC 3.2.1.23), beta-glucosidase (EC 3.2.1.21) and alpha-glucosidase (EC 3.2.1.20) activities whereas beta-glucuronidase (EC 3.2.1.31) was lowered. Compared with the control group, FOS and TOS diets led to a significant increase in H2 and CH4 excretion; the GOS diet increased only CH4. Analysis of caecal contents revealed a decrease in pH for all diets compared with controls. Total short-chain fatty acid (SCFA) concentration increased significantly in all groups, but the SCFA profile differed between treatment groups. It was concluded that the three oligosaccharides studied had different effects which may be linked to their chemical structure. Some of these effects may be beneficial to human health.

Animals↗
PubMed2002

Oligosaccharides in infant formula.

Breast-feeding is the golden standard for infant feeding. However, the majority of a few week old infants are fed with a second choice infant feeding, cow's milk based formula. Amongst the multiple differences between human and cow's milk regards the development of the gastro-intestinal flora: the flora of the breast-fed infant being richer in bifidobacteria and lactobacilli. Both species are known to be potentially beneficial for the health of the host. The absence of oligosaccharides, the third largest component in human milk, in cow's milk is likely to account for the differences in colonic flora. The oligosaccharide content and concentration in breast milk is - just as for the other macronutrients - a dynamic process, making it impossible for industry to mimic nature. However, if the composition cannot be mimicked, the effect and function can be imitated. The addition of two oligosaccharides, galacto-oligosaccharides and inulin, to cow's milk based infant formula has been shown to have a bifidogenic effect, and to stimulate the growth of bifidi and lactobacilli. In conclusion, the addition of oligosaccharides to cow's milk based infant formula brings this alternative, second choice infant feeding one step closer to the golden standard of human milk. But, prolonged breast-feeding should still be promoted with maximum effort.

Animals↗
PubMed1999

Secretion of 13C-labelled oligosaccharides into human milk and infant's urine after an oral [13C]galactose load.

Human milk oligosaccharides seem to play an important role in the infant's defense against bacterial and viral infections of the gastrointestinal and the urogenital tract. In this study, we investigated the influence of dietary carbohydrates on the biosynthesis of lactose and oligosaccharides in the human mammary gland and their renal excretion by the human milk-fed infant. For this purpose, a lactating woman was given 27 g galactose (Gal) containing 2 g [13C] Gal (1-13C/99%) immediately after breakfast. In the following 36 h, milk (5-10 ml) was collected before each nursing. Infant's urine was collected over a period of 24 h. 13C-enrichment was measured in total milk, milk fat and protein, in the carbohydrate fraction as well as in urine by isotope ratio mass spectrometry (IRMS). Milk carbohydrates and deproteinized urine samples were fractionated by Sephadex G25 gel filtration and further analyzed by IRMS, high performance thin layer chromatography and and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). IRMS revealed that in milk a maximal delta 13CPDB was reached within 8 h after Gal intake which then rapidly declined in the following 8 h. The cumulative 13C-elimination over this first peak was 6.9% of the oral 13C-dose. The highest 13C-enrichment was detectable in the carbohydrate fraction, mainly in lactose and neutral oligosaccharides. Compared to the enrichment of human milk, the delta 13CPDB of infant's urine was delayed. In urine, the highest amount of 13C was found in the Sephadex G25 fractions which mainly contained lactose, fucosyl-lactose, lacto-N-tetraose (LNT), fucosyl-LNT and difucosyl-LNT. For further characterization, individual components were separated by HPAEC-PAD and subsequently analyzed by fast atom bombardment mass spectrometry and IRMS. The data show, that orally applied Gal is incorporated in milk, especially in lactose and neutral oligosaccharides. Obviously, some of these components were absorbed by the infant and then excreted with urine. There, oligosaccharides may serve as analogous receptors for bacterial or viral adhesion molecules, and, hence, may prevent urogenital infections in breastfed infants.

Administration, Oral↗
PubMed2000

Massive in vitro synthesis of tagged oligosaccharides in 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside treated HT-29 cells.

Permanent exposure of differentiated HT-29 cells to the sugar analogue, 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha- O -bn) leads to marked effects upon the phenotypic properties of mucin-secreting or enterocyte-like HT-29 cells: an inhibition in the secretion of mucins, a blockade in the apical targeting of membrane brush border glycoproteins and a swelling of cells with intracellular accumulation of numerous vesicles. Folch extraction and partition of treated enterocyte-like HT-29 cells revealed a very important accumulation of orcinol and/or resorcinol reactive material in the upper phase (usually containing gangliosides), as compared with untreated HT-29 cells and with treated and untreated Caco-2 cells. Structural analysis indicated the accumulation of a series of GalNAcalpha- O -bn derived oligosaccharides, most of them with the common core Galbeta1-3GalNAcalpha- O -bn. These oligosaccharides contained residues of GlcNAc, Gal, Neu5Ac, or Fuc. In particular, the tagged sialyl-Lewis(x)was identified, as well as more complex sialylated derivatives, including the sialyl-Lewis(x)substituted by an additional Neu5Ac residue. The benzylated oligosaccharides were not significantly detected in the culture medium except for Galbeta1-3GalNAcalpha- O -bn. Upon reversion of the treatment, these derivatives dis-appeared from the cells within few days, however were not recovered as such in the culture medium. Intracellular degradation occurred with desialylation and defucosylation as the first steps. The spectacular accumulation of benzylated oligosaccharides in HT-29 cell, permanently exposed to GalNAcalpha- O -bn very likely plays an important role in the alterations of cellular processes previously described in this cell line. The HT-29 cell culture system also appears to be an efficient source of several tagged oligosaccharides.

Cell Differentiation↗
PubMed2001

Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells.

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.

Adenocarcinoma↗
PubMed2001

N-linked oligosaccharides of cobra venom factor contain novel alpha(1-3)galactosylated Le(x) structures.

Cobra venom factor (CVF), a nontoxic, complement-activating glycoprotein in cobra venom, is a functional analog of mammalian complement component C3b. The carbohydrate moiety of CVF consists exclusively of N-linked oligosaccharides with terminal alpha1-3-linked galactosyl residues, which are antigenic in human. CVF has potential for several medical applications, including targeted cell killing and complement depletion. Here, we report a detailed structural analysis of the oligosaccharides of CVF. The structures of the oligosaccharides were determined by lectin affinity chromatography, antibody affinity blotting, compositional and methylation analyses, and high-resolution (1)H-NMR spectroscopy. Approximately 80% of the oligosaccharides are diantennary complex-type, approximately 12% are tri- and tetra-antennary complex-type, and approximately 8% are oligomannose type structures. The majority of the complex-type oligosaccharides terminate in Galalpha1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1, a unique carbohydrate structural feature abundantly present in the glycoproteins of cobra venom.

Carbohydrate Sequence↗
PubMed1992

Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.

Amidohydrolases↗
PubMed1992

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.

Acetylgalactosamine↗
PubMed1994

Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells.

Recombinant human erythropoietin (rHuEPO) is used abundantly in the clinic to stimulate red blood cell growth in anaemic patients. The efficacy of the drug depends strongly on the extent of sialylation of its carbohydrate moiety. Prompted by conflicting literature reports on the issue, we reinvestigated the structures of the intact sialylated carbohydrate chains of rHuEPO expressed in Chinese hamster ovary (CHO) cells. The asparagine-linked oligosaccharides were released from rHuEPO with N-glycanase and fractionated by anion-exchange chromatography. The O-linked oligosaccharides were released under alkaline borohydride conditions. The primary structures of the major sialylated N- and O-type oligosaccharides were identified by 500-MHz 1H-NMR spectroscopy, supported by data from composition analysis, methylation analysis, low- and high-pH anion-exchange chromatography, and fast atom bombardment-mass spectrometry. The most abundant N-linked oligosaccharides in CHO cell-derived rHuEPO were found to be di-antennary, 2,4-branched tri-antennary, 2,6-branched tri-antennary and tetra-antennary chains (in the ratio of 7:6:5:82), with the latter containing between zero and three repeating N-acetyllactosamine units, in well-defined branches. The major (> 95%) di-, tri- and tetra-antennary structures are fully sialylated, i.e. they have two, three and four sialic acid residues, respectively, linked exclusively alpha(2-->3) to galactose residues. The majority (> 95%) of N-linked structures contain alpha(1-->6)-linked fucose at the proximal GlcNAc residue. The O-type mono- and disialyl oligosaccharides were characterized as a linear tri- and a branched tetra-saccharide, respectively.

Amidohydrolases↗
PubMed1996

Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis.

The major diagnostic antigen of Paracoccidioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain. This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence. It contains a neutral high-mannose core (Man7GlcNAc2) to which a (1-->6)-linked alpha-D-Manp chain of variable length, substituted at the 2-O positions by single alpha-D-Manp residues, is attached. A terminal unit of beta-D-galactofuranose is (1-->6)-linked to one of the (1-->2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide. The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the beta-galactofuranosyl unit. The complete structure was determined by methylation analysis, 1H-NMR, mass spectrometry, acetolysis and mannosidase degradation. Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex18GlcNAc2 to Hex10GlcNAc2 which accounts for the diffuse migration of the gp43 in polyacrylamide gels. The average size of the most frequent subtype is Hex13.6GlcNAc2. Dilute acid treatment to remove beta-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.

Acetylation↗
PubMed1997

Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface.

The mechanism underlying molecular recognition of lectins was elucidated by a novel solid phase binding assay system based on surface plasmon resonance. When the apparent affinities of interactions between chitooligosaccharides and wheat germ agglutinin were compared between lectin-immobilized and oligosaccharide-immobilized assay systems, the affinity constants (Ka) calculated for the former system were in good agreement with the previously reported values measured in solution. On the other hand, in the latter system, the calculated Ka could be more than 10,000 times higher than the values in solution at lower lectin concentrations. To elucidate the reason for this, we systematically investigated the effects of the oligosaccharide immobilized density and the lectin valence on the apparent affinity in the oligosaccharide-immobilized assay system. Both the apparent association (k[ass]) and dissociation rate constants (k[diss]) showed a tendency to decrease as the oligosaccharide density increased. This effect was most remarkable for the interaction possessing an extremely fast intrinsic k(ass). Oligomerization of lectin enhanced the avidity due to a significant reduction in k(diss). These phenomena could be explained by considering the nonhomogeneous conditions under which binding occurred. The reaction in a nonhomogeneous state is limited by the mass transport effect, and the effect of rebinding becomes so large that it cannot be disregarded. These findings are the first to demonstrate the importance of the mass transport effect in modulating the affinity of lectin for oligosaccharides on a solid phase surface.

Amino Sugars↗
PubMed1998

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells.

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N -acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.

Acetylglucosamine↗
PubMed1998

Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo.

In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase. It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway. To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins. These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains. Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S. cerevisiae.

Animals↗