Release Of Oligosaccharides Research Articles

5] Release of oligosaccharides from glycoproteins by hydrazinolysis

Hydrazinolysis is an effective technique for the release of unreduced O-linked oligosaccharides from glycoproteins. The mechanism leading to the release of O-linked oligosaccharides has not yet been investigated, although β elimination is suspected. Reaction conditions under which N- and O-linked oligosaccharides can be released from glycoproteins have been established empirically after investigation of the release of such oligosaccharides from a range of glycoprotein standards, whose glycosylation characteristics had been previously well defined. Despite the structural diversity of oligosaccharides and protein moieties associated with these standards, similar reaction conditions are found to allow essentially complete release of unreduced N- and O-linked oligosaccharides. Selective release of O-linked oligosaccharides with small release of N-linked oligosaccharides can be achieved with mild hydrazinolysis reaction conditions. Hydrazinolysis can therefore be used in three different ways, namely—to release all (N- and O-linked) oligosaccharides from a glycoprotein, to release selectively just O-linked oligosaccharides, and to release sequentially first O-linked, and then N-linked oligosaccharides.

Structural profiling of oligosaccharides of glycoproteins.

AbstractThe release of oligosaccharides from glycoproteins provides useful information on the type and extent of glycosylation that can be correlated with the effect of glycosylation on the function of the glycoprotein. However, to fully characterize the diversity of oligosaccharide structures within a glycoprotein it is necessary to assign each oligosaccharide structure to a particular glycosylation site (i.e., the site-specific glycosylation pattern). Only when this has been done can accurate models be made of glycoproteins to study the roles of specific oligosaccharides. The complete characterization of the site-specific glycosylation patterns of a glycoprotein requires the fulfillment of three conditions, namely: 1. The isolation of glycopeptides with only one glycosylation site. 2. The identification of the glycosylation site relative to the amino acid sequence of the protein. 3. Characterization of the structures of the oligosaccharide chains and the nature of the carbohydrate-protein linkage at each site. KeywordsGlycosylation SiteIodoacetic AcidPorous Graphitized CarbonOligosaccharide StructurePhenyl Methyl SiliconeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Separation of neutral oligosaccharide alditols from human meconium using high-pH anion-exchange chromatography.

Neutral reduced oligosaccharides are in general not sufficiently retained to achieve adequate separation and reproducible chromatography using high-pH anion-exchange chromatography. We describe a method to increase the retention using two columns in series. This method has been applied to the separation of oligosaccharides purified from human meconium glycoproteins, obtained as their alditols after alkaline-borohydride release of oligosaccharides. The neutral oligosaccharide alditols were significantly retained upon two CarboPac PA-100 columns, connected in series, and eluted in 80 mM sodium hydroxide between 4 and 10 min. Three sialylated alditols studied were substantially retained and could be eluted in a gradient of 0-500 mM sodium acetate-80 mM sodium hydroxide between 10 and 45 min. The elution patterns were based on their size, charge and linkage, such that oligosaccharide alditol isomers could be separated.

Effect of cell attachment and growth on the synthesis and fate of dolichol-linked oligosaccharides in Chinese hamster ovary cells.

The inhibition of cellular processes in suspended anchorage-dependent Chinese hamster ovary (CHO) cell lines and their restoration upon attachment to a solid substrate has been used as a model to study the relationship between oligosaccharide-diphospho-dolichols and their metabolic products (glycoprotein and soluble oligosaccharide material, i.e. oligosaccharide phosphates and neutral oligosaccharides). Using metabolic labelling we demonstrated that suspended cells have a low incorporation rate into lipid intermediates and into glycoproteins. The oligosaccharide-lipid populations are mainly glucosylated and the neutral oligosaccharides have exclusively a chitobiosyl residue at their reducing end. In contrast, monolayer cells exhibit a high incorporation rate into lipid intermediates with a pattern dominated by two species containing either two or five mannose residues, and into glycoproteins with a pattern similar to the one observed for suspended cells (i.e. glucosylated species). In monolayer cells the neutral oligosaccharides possess either one or two GlcNAc residues at their reducing end. The variations in the nature and in the quantity of soluble oligosaccharide material as a function of the cell density reflects regulatory points in the synthesis of N-glycosyl proteins. The first regulatory point could be the control of the quantity of non-glucosylated oligosaccharide-lipids to be channelled toward the glucosylated lipid-donor pool. The level of this donor pool being constant, the oligosaccharide-transferase could utilize oligosaccharide-lipid donors at a constant rate by two different reactions: either transfer onto protein when acceptors are available, or transfer onto water generating neutral oligosaccharides possessing two GlcNAc residues at the reducing end. Another regulatory point would be the degradation of a part of neoglycoproteins leading to the release of neutral oligosaccharides possessing one GlcNAc residue at the reducing end.

S20.12 Analysis of protein portion of porcine gastric mucus glycoprotein after release of O-Linked oligosaccharide by gas-phase hydrazinolysis

S20.12 Analysis of protein portion of porcine gastric mucus glycoprotein after release of O-Linked oligosaccharide by gas-phase hydrazinolysis

Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown.

A chitinase, purified to homogeneity from ethylene-treated bean leaves, was applied to actively growing mycelial cells of Rhizoctonia solani to evaluate a potential antifungal activity. Light microscopic investigations at 30-min intervals following enzyme exposure revealed the induction of morphological changes such as swelling of hyphal tips and hyphal distortions. More precise information concerning fungal cell alteration was obtained by ultrastructural observation and cytochemical detection of chitin distribution in fungal cell walls. Chitin breakdown was found to be an early event preceding wall disruption and cytoplasm leakage. The large amounts of chitin present in the walls of control R. solani cells and the rapid chitin hydrolysis upon chitinase treatment lead us to suggest that this polysaccharide is one of the main components of this fungal cell wall and is readily accessible to chitinase, especially in the apical zone. By 60 min after enzyme treatment, labeled molecules were observed in the vicinity of some fungal cells, suggesting the release of chitin oligosaccharides from fungal cell walls. The antifungal activity of the bean chitinase on cells of R. solani grown in culture is discussed in relation to the potential of genetically modified transgenic plants to resist attack by R. solani through an antimicrobial activity in planta.

A study of possible sulfate loss during the chemical release of sulfated oligosaccharides from glycoproteins

A study of possible sulfate loss during the chemical release of sulfated oligosaccharides from glycoproteins

A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.

A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.

Different fates of the oligosaccharide moieties of lipid intermediates

We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides.

Characterization of a sialic‐acid‐rich mucus glycoprotein secreted by a premalignant human colorectal adenoma cell line

The human colonic cell line PC/AA, derived from an adenoma, retains in vitro colonic cell differentiation, notably the production of mucus glycoproteins. The PC/AA adenoma cells produce an extracellular gel layer in culture. The PC/AA gel could be isolated by extraction of the cell cultures with guanidine hydrochloride. The extracted material was purified by gel filtration and caesium chloride density-gradient centrifugation and showed properties typical of mucus glycoproteins, namely, a carbohydrate content above 60% of dry weight rich in N-acetylgalactosamine and sialic acid and low in mannose; an amino acid composition with high serine threonine and proline content; a molecular weight above 1,000 kDa on Sepharose CL 4B chromatography and on SDS-polyacrylamide gel electrophoresis under reducing conditions (greater than 200 kDa); a buoyant density of approximately 1.48 g/ml and the release of oligosaccharides by the alkaline beta-elimination reaction. Comparison of the gel mucus glycoprotein purified from premalignant PC/AA cells with normal human colon mucin showed that it has a higher sialic acid content. This suggests that higher sialic acid levels may precede the development of malignancy.

Carbohydrate analysis of glycoproteins A review

Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. As little as five years ago, glycosylation was believed to play no significant role in the function of glycoproteins. Recent large scale testing of glycoprotein-based pharmaceuticals has indicated that both the extent and type of glycosylation can play a central role in glycoprotein activity. Although methods for compositional and sequence analysis of proteins and nucleic acids are generally available, similar methods have yet to be developed for carbohydrate oligomers and polymers. This review focuses on new, developing methods for the analysis and sequencing of the carbohydrate portion of glycoproteins. Included are: (1) the release of oligosaccharides and hydrolysis of carbohydrate chains using enzymatic and chemical methods; (2) fractionation by LPLC, electrophoresis, HPLC, and lectin affinity chromatography; (3) detection through the preparation of derivatives or by new electrochemical methods; (4) analysis by spectroscopic methods, including MS and high-field NMR; and (5) their sequencing through the use of multiple, well-integrated techniques. The ultimate goal of the analytical approaches discussed is to firmly establish structure and, thus, permit the study of structure-function relationships and eventually to allow the intelligent application of carbohydrate remodeling techniques in the preparation of new glycoproteins.

Release of oligosaccharides possessing reducing-end N-acetylgalactosamine from mucus glycoprotein in [formula omitted] sp. OH-11242 culture medium through action of endo-type glycosidase

A crude enzyme preparation from a culture medium of Streptomyces sp. OH-11242 contained endo-α-N-acetylgalactosaminidase activity. The activity could be induced by the addition of purified porcine gastric mucin to the culture medium. Oligosaccharides corresponding to approximately 2–14 glucose units were detected in the culture medium and also in an incubated reaction mixture of crude enzyme preparation and mucus glycoprotein. The resulting product with N-acetylgalactosamine at the reducing terminal implied the presence of a new type of endo-glycosidase liberating not only Ga1ß1–3Ga1NAc but also other larger oligosaccharides by hydrolysis of the O-glycosidic linkage between Ga1NAc and Ser (Thr).

Estrogen preferentially stimulates lactosaminoglycan-containing oligosaccharide synthesis in mouse uteri.

The effects of steroid hormones on the synthesis of lactosaminoglycan (LAG)-containing oligosaccharides by mouse uteri are reported. The uterine LAG-containing oligosaccharides were degraded partially by Pseudomonas endo-beta-galactosidase, releasing an oligosaccharide of the apparent structure: Gal beta----N-acetylglucosaminyl(----N-acetylgalactosaminyl)beta 1,3----galactose. A larger fraction of the LAG-containing oligosaccharides bound to pokeweed mitogen than to Datura stramonium lectin, suggesting the presence of highly branched structures. LAG-containing oligosaccharides were resistant to sequential digestion with Pronase, nitrous acid, hyaluronidase, and chondroitinase ABC. These polysaccharides exhibited a Gal:GlcNAc:GalNAc ratio of approximately 1.0:1.0:0.3 and were not fucosylated. The ion-exchange behavior of the LAG-containing oligosaccharides before and after mild acid hydrolysis indicated the presence of sialic acid residues. The LAG-containing glycopeptides were highly resistant to beta-elimination but were released quantitatively by hydrazinolysis, demonstrating an N-linkage to protein. Binding to pokeweed mitogen was markedly enhanced following release of these oligosaccharides from peptides by hydrazinolysis, suggesting that peptide-bound oligosaccharides were partially inaccessible to the lectin. Molecular exclusion chromatography of the oligosaccharides released by hydrazinolysis revealed a broad distribution ranging from Mr 4,000 to 15,000 with a median Mr of approximately 8,000. We extended the above observations by determining how the steroid hormones 17-beta-estradiol (E2) and progesterone affected synthesis of the LAG-containing oligosaccharides in ovariectomized mice. Generally, E2 and a number of E2 agonists stimulated glycoconjugate synthesis; however, chronic E2 treatment or combined treatment with E2 plus progesterone caused the synthesis of most glycosaminoglycans to return to basal levels. In contrast, E2 either alone or in combination with progesterone stimulated synthesis of LAG-containing oligosaccharides in preference not only to glycosaminoglycans but also to other classes of N-linked oligosaccharides. This effect was apparent during both priming and nidatory E2 treatments. Collectively, these data provide the first demonstration of LAG-containing oligosaccharides in uteri and for the hormonally regulated synthesis of lactosaminoglycans. In addition, this is the first demonstration of the ability of steroid hormones to induce the synthesis of certain types of N-linked oligosaccharides in preference to others in the same tissue.

Pathway of assimilate transfer between mesophyll cells and minor veins in leaves of Cucumis melo L.

Photoassimilating mature leaves of Cucumis melo exported carbon at a rate of 1.7 mg C·dm(-2)·h(-1). Radiolabeling with (14)C showed that stachyose and raffinose are the main carbohydrates translocated. Autoradiograms indicated that sieve elements of the abaxial phloem of minor veins are the sole conduits for carbon export from mature leaves and carbon import into immature leaflets. Sieve elements of the abaxial phloem are associated with intermediary cells which are intimately connected with the surrounding mesophyll cells by numerous plasmodesmata. Photoassimilate, labeled with (14)C, was released into the leaf apoplast and could be trapped in a buffer solution circulating over the abraded adaxial epidermis. Carbon efflux was 1% of the carbon-export rate. A comparable distribution of (14)C among the sugars, amino acids and organic acids, recovered from the free space and from leaf extracts, was recorded. The composition of released (14)C-labeled carbohydrates in the free space resembled the pattern of photoassimilate, but differed clearly from the translocate. Release of organic compounds into the leaf apoplast was stimulated by chelating agents like Na-ATP, ethylenediaminetetraacetic acid and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; a correlation between carbon efflux into the apoplast and carbon export from the leaf was not detected. It is suggested that the release of organic compounds into the leaf apoplast of Cucumis melo is the consequence of a general leakage from mesophyll and vascular parenchyma cells. A selective release of transport oligosaccharides was not observed. The experimental results presented here do not preclude a symplastic transfer of assimilates in mature leaves.

The release of oligosaccharides from glycoproteins by endo- β- N-acetylglucosaminidase of Flavobacterium sp.

Endo- β- N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage.

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.

Structural changes in membranes of large unilamellar vesicles after binding of sodium cholate

The interaction of the bile salt cholate with unilamellar vesicles was studied. At low cholate content, equilibrium binding measurements with egg yolk lecithin membranes suggest that cholate binds to the outer vesicle leaflet. At increasing concentrations, further bile salt binding to the membrane is hampered. Before the onset of membrane solubilization, diphenylhexatriene fluorescence anisotropy decreases to a shallow minimum. It then increases to the initial value in the cholate concentration range of membrane solubilization. At still higher cholate concentrations, a drop in fluorescence anisotropy indicates the transformation of mixed disk micelles into spherical micelles. Perturbation of the vesicle membranes at molar ratios of bound cholate/lecithin exceeding 0.15 leads to a transient release of oligosaccharides from intravesicular space. The cholate concentrations required to induce the release depend on the size of the entrapped sugars. Cholesterol stabilizes the membrane, whereas, in spite of enhanced membrane order, sphingomyelin destabilizes the membrane against cholate. Freeze-fracture electron microscopy and phosphorus-31 nuclear magnetic resonance (31P NMR) also reflect a change in membrane structure at maximal cholate binding to the vesicles. In 31P NMR spectra, superimposed on the anisotropic line typically found in phospholipid bilayers, an isotropic peak was found. This signal is most probably due to the formation of smaller vesicles after addition of cholate. The results were discussed with respect to bile salt/membrane interactions in the liver cell. It is concluded that vesicular bile salt transport in the cytoplasm is unlikely and that cholate binding is restricted to the outer leaflet of the canalicular part of the plasma membrane.

Autolytic Release of Mannoproteins from Cell Walls of Saccharomyces cerevisiae

Summary: Autolysis of purified Saccharomyces cerevisiae cell walls resulted in the release of several components thought to be mannoprotein in nature, since they were retained by Concanavalin A-Sepharose. The most abundant was a 29 kDal molecule which was also a major species among mannoproteins solubilized by the β-glucanase complex Zymolyase. There was a concomitant release of glucose and mannose oligosaccharides and of β-glucanase activity. Glucanase and protease inhibitory treatments considerably lowered the release of mannoproteins. The use of different protease inhibitors during autolytic incubations interfered with at least two proteases (one of them being a cysteine protease) apparently involved in the solubilization and partial degradation of mannoproteins from the walls.

The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase.

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.

G.L.C. of O-methyloxime and alditol acetate derivatives of neutral sugars, hexosamines, and sialic acids: “One-pot” quantitative determination of the carbohydrate constituents of glycoproteins and a study of the selectivity of alkaline borohydride reductions

A new procedure for the quantification by g.l.c. of the carbohydrate constituents of glycoproteins is proposed which involves ( a) simultaneous action of neuraminidase and neuraminic acid aldolase, ( b) hydrolysis with 4 m trifluoroacetic acid at 125° for 1 h, and ( c) conversion of the products into O-methyloxime acetates and g.l.c. The procedure has been successfully tested on fetuin, transferrin, α 1-acid glycoprotein, and mucin. The g.l.c. conditions used also enabled the complete separation of O-methyloxime and alditol acetate derivatives in one run, so that the release of carbohydrate chains from glycoproteins by treatment with alkaline borohydride can be investigated conveniently. There was complete release of O-linked oligosaccharides from fetuin on treatment with 0.1 m NaOH/0.8 m NaBH 4 (68 h, 37°) or 0.05 m KOH/ m KBH 4 (24 h, 45°) and also release of ∼75% and 35–40%, respectively, of N-asparagine-linked chains. Reduced oligosaccharides were formed only from O-linked chains; the mechanism by which N-linked chains were released is still not clear.