Studies on the asymmetrically attached oligosaccharide of rabbit immunoglobulin-G. II. On the biological function of the C2-oligosaccharide.
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Animals↗
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[On the cleavage and resorption of oligosaccharides in the small intestine. 3. Importance of glucosidic enzymes in the decomposition of malto-oligosaccharides and starch].
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Amylases↗
[On splitting and resorption of oligosaccharides in the small intestine. II. Formation of glucose in tissue slices of rat intestine from malto-oligosaccharides].
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Amylases↗
Chemometrics of oligosaccharide analysis. Parameterization of HPLC of PA-oligosaccharides.
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Animals↗
"Cap-Tag"-Novel Methods for the Rapid Purification of Oligosaccharides Prepared by Automated Solid-Phase Synthesis Financial support from the donors of the Petroleum Research Fund, administered by the ACS (ACS-PRF 34649-G1), Merck (Predoctoral Fellowship for E.R.P.), Boehringer-Ingelheim (Predoctoral Fellowship for E.R.P.), and the NIH (Biotechnology Training Grant for M.C.H.) is gratefully acknowledged. Funding for the MIT-DCIF Inova 501 was provided by the NSF (Award CHE-9808061). Funding for the MIT-DCIF Avance (DPX) 400 was provided by the NIH (Award 1S10RR13886-01). We thank Silicycle (Quebec City, Canada) for the generous gift of fluorous silica gel and isocyanate silica gel scavenger resin. We thank Dr. O. Plante for helpful discussions and help with the automated oligosaccharide synthesizer.
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Journal Article↗
Enzymatic transfer of mannose from mannosyl-phosphoryl-polyprenol to lipid-linked oligosaccharides by pig aorta.
A particulate enzyme preparation prepared from the intimal layer of pig aorta catalyzed the transfer of mannose from mannosyl-phosphoryl-polyprenol (MPP) into a series of oligosaccharides that were linked to lipid. The reaction required detergent with Triton X-100 and NP-40 being best at a concentration of 0.5%. Several other detergents were inactive or only slightly active. The pH optima for this activity was about 7 to 7.5 in Tris buffer and the apparent Km for MPP was about 2 x 10(-7) M. The reaction was not stimulated by the addition of divalent cation and, in fact, was inhibited by the high concentrations of cation. The addition of EDTA did not inhibit the transfer of mannose from MPP and was somewhat stimulatory. The transferase(s) activity was "solubilized" from the particles by treatment with Triton X-100. This solubilized enzyme still formed a series of lipid-linked oligosaccharides from either MPP or GDP-mannose. The oligosaccharides were released from the lipid by mild acid hydrolysis and were separated by paper chromatography. Some five or six radioactive oligosaccharides were formed from either MPP or from GDP-mannose and these oligosaccharides had similar mobilities upon paper chromatography. However, MPP was a better donor for the larger oligosaccharides (i.e. those containing 8, 9, or 10 sugar residues), whereas GDP-mannose was better for formation of the oligosaccharide containing 7 sugar residues. In the presence of EDTA and detergent no MPP was formed from GDP-mannose, but radioactivity was still incorporated into the lipid-linked oligosaccharides. Under these conditions essentially all of the radioactivity was in the oligosaccharide containing 7 sugar residues. Since much of this activity could be released as mannose by acetolysis, GDP-mannose may be the direct mannosyl donor for formation of 1 leads to 6 branches. Oligosaccharides 7, 8, 9, and 10 were isolated and partially characterized in terms of their molecular weights, sugar composition, susceptibility to alpha-mannosidase, and 14C products formed by acetolysis and periodate oxidation. The molecular weights ranged from 1310 for oligosaccharide 7 to 1750 for oligosaccharide 10. Hydrolysis of each oligosaccharide and reduction with NaB3H4 gave the expected ratio of [3H]hexitol to [3H]hexosaminitol based on the molecular weight of the oligosaccharide. However, the hexitol fraction contained [3H]mannitol and [3H]glucitol. Since the amount of radioactivity in glucitol was 2 to 4 times that in mannitol and since only glucosaminitol was found in the amino sugar peak, it seems likely that each 14C-oligosaccharide was contaminated with an unlabeled oligosaccharide of equal molecular weight containing glucose and GlcNAc. Acetolysis of the 14C-oligosaccharides gave rise to 14C peaks of mannose, mannobiose, and mannotriose. In the larger oligosaccharides, most of the radioactivity was in mannobiose whereas in oligosaccharide 7 most of the radioactivity was in mannose...
Animals↗
Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica.
The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242] to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor. The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by high-resolution proton NMR. Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N-acetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal alpha-galactose residue(s) linked to C3 of beta-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Gal beta 1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.
Animals↗
Acidic oligosaccharides from pectin hydrolysate as new component for infant formulae: effect on intestinal flora, stool characteristics, and pH.
OBJECTIVES: To come even closer to the functional composition of human milk, acidic oligosaccharides (AOS) from pectin were added to well known neutral prebiotics (galacto-oligosaccharides (GOS) and long-chain fructo-oligosaccharides (FOS)). The effect of AOS and GOS/FOS/AOS on intestinal flora, stool characteristics as well as acceptance and tolerance was investigated. METHODS: Human milk contains 75% to 85% neutral and 15% to 25% acidic oligosaccharides. In this prospective, randomized, double blind study, a mixture of 80% neutral oligosaccharides (from long-chain galacto- and long-chain fructo-oligosaccharides) with 20% acidic oligosaccharides derived from pectin hydrolysis was investigated. Forty-six term infants were fed a standard formula supplemented with either maltodextrin as control (n=15), or with 0.2 g acidic oligosaccharides (n=16), or with the latter plus 0.6 g neutral oligosaccharides (mixture of galacto- and fructo-oligosaccharides; n=15). Fecal flora using plating technique and pH were measured. Stool characteristics and possible side effects (crying, vomiting, and regurgitation) were recorded. RESULTS: There was no difference in the bifidobacteria counts between the control and the group supplemented with acidic oligosaccharides alone (8.75+/-0.50 vs. 8.58+/-0.94 log colony forming units [CFU]/g stool). In infants fed the combination of acidic and neutral oligosaccharides, bifidobacteria were increased (9.61+/-0.70 log CFU/g stool; P<0.01). The same pattern was observed with lactobacilli. Stool consistency was softest in infants fed the complete oligosaccharide mixture, but also in those fed formula supplemented with acidic oligosaccharides alone, the stool consistency was significantly softer compared with the control group. Fecal pH increased in the controls, remained constant in acidic oligosaccharides alone, and decreased in the complete mixture of oligosaccharides group. CONCLUSION: There was no difference in growth, crying, vomiting, and regurgitation patterns between the groups. In summary, acidic oligosaccharides from pectin hydrolysate are well tolerated as ingredient in infant formulae but do not affect intestinal microecology.
Bifidobacterium↗
The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.
Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.
Acetamides↗
Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2.
It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series--series 1: oligosaccharides processed from Manalpha1-2Manalpha1-6 (Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3) Manbeta1-4GlcNAc (M9A') and Manalpha1-2Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Manalpha1-6(Manalpha1- 2Manalpha1-3)Manbeta1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.
Acetylglucosamine↗
Cell type and maturation stage-dependent polymorphism of N-linked oligosaccharides on murine lymphocytes and lymphoma cells.
We analyzed the overall structures of N-linked oligosaccharides on glycoproteins of various murine lymphocytic and lymphoma cells employing a newly developed method which was performed on high-performance liquid chromatography after derivatization of oligosaccharides with 2-aminopyridine. A total of 15 types of bi, tri- and tetra-antennary N-acetyllactosamine-type oligosaccharides with or without fucose and oligomannose-type oligosaccharides were identified on these cells in variable amounts depending on the type and maturation stage of the cells. It was found that all murine lymphocytic cells carry N-acetyllactosamine-type oligosaccharides with the additional alpha-linked galactose residue on the non-reducing ends. Thymocytes had exceptionally large amounts of oligosaccharides with one or even two alpha-galactose residues per molecule. In contrast, peripheral resting T cells possessed those oligosaccharides only in a small amount, although the cells produced more the oligosaccharides after stimulation with Con A. Two thymoma lines such as BW 5147 and EL-4 and one B cell lymphoma line WEHI231 contained relatively large amount of oligosaccharides with alpha-galactose residues. Significant change of the molar ratio of component carbohydrates by cell activation was observed also in oligommanose-type oligosaccharides which were few in resting T cells but were markedly increased in Con A activated cells. Molar ratio of triantennary oligosaccharides in total N-acetyllactosamine type oligosaccharides was high in thymocytes and low in resting T cells, but was increased in T cells after Con A activation. It was also very high in WEHI 231 B cell lymphoma. Although BW 5147 and EL-4 thymoma did not contain tri-antennary oligosaccharides in high proportion, they carried larger tetra-antennary oligosaccharides with an N-acetyllactosamine repeating unit in definitive amounts. It is suggested from these results that overall structures of oligosaccharides on cell surface proteins of lymphocytes are finely controlled with link to cell differentiation, activation and transformation.
Animals↗
Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein.
The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Preparation and structural characterization of large heparin-derived oligosaccharides.
Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (< 5000) and high-molecular-weight (> 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.
Animals↗
Biosynthesis of the core region of yeast mannoproteins. Formation of a glucosylated dolichol-bound oligosaccharide precursor, its transfer to protein and subsequent modification.
A new membrane preparation from Saccharomyces cerevisiae was developed, which effectively catalyzes the synthesis of large oligosaccharide-lipids from GDP-Man and UDP-Glc allowing a detailed study of their formation and size. The oligosaccharide from an incubation with GDP-Man could be separated by gel filtration chromatography into several species consisting of two N-acetylglucosamine (GlcNAc) residues at the reducing end and differing by one mannos unit; the major compound formed has the composition (Man)9(GlcNAc)2. Upon incubation with UDP-Glc, three oligosaccharides corresponding to the size of (Glc)1-3(Man)9(GlcNAc)2 are formed. Thus, the oligosaccharides generated in vitro by the yeast membranes appear to be identical in size with the oligosaccharides found in animal systems. In addition the results indicate that dolichyl phosphate mannoe (DolP-Man) is the immediate donor in assembling the oligosaccharide moiety from (Man)5(GlcNAc)2 to (Man)9(GlcNAc)2. All three glucose residues are transferred from DolP-Glc. Experiments with isolated [Glc-14C]oligosaccharide-lipid as substrate demonstrated that the oligosaccharide chain is transferred to an endogenous membrane protein acceptor. Moreover, transfer is followed by an enzymic removal of glucose residues, due to a glucosidase activity associated with the membranes. Glucose release from the free [Glc-14C]oligosaccharide is less effective than from protein-bound oligosaccharide. Glycosylation was also observed using [Man-14C]oligosaccharide-lipid or DolPP-(GlcNAc)2 as donor. However, transfer in the presence of glucose seems to be more rapid. The mannose-containing oligosaccharide, released from the lipid, was shown to function as a substrate for further chain elongation reactions utilizing GDP-Man but not DolPP-Man as donor. It is suggested that the immediate precursor in the synthesis of the heterogeneous core region, (Man)12-17(GlcNAc)2, of yeast mannoproteins is a glucose-containing lipid-oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2, i.e. only part of what has been defined as inner core is built up on the lipid carrier. After transfer to protein the oligosaccharide is modified by excision of the glucose residues, followed subsequently by further elongation from GDP-Man to give the size of th oligosaccharide chains found in native mannoproteins.
Diterpenes↗
Formation of unusual mannosamine-containing lipid-linked oligosaccharides in Madin-Darby canine kidney cell cultures.
Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol is the major lipid-linked oligosaccharide (LLO) produced by Madin-Darby canine kidney cells in culture. However, when these cells are incubated in the presence of millimolar concentrations of mannosamine and labeled with [2-3H]mannose, they accumulate various LLO that have smaller-sized oligosaccharides with unusual structures and the Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol is not detected. Thus in the presence of 10 mM mannosamine, more than 80% of the oligosaccharides are eluted from concanavalin A-Sepharose with 10 mM alpha-methylglucoside, indicating that they no longer have the tight-binding characteristics of control oligosaccharides. In addition, 20-40% of these oligosaccharides bind to Dowex 50-H+, indicating the presence of mannosamine in these structures. Interestingly enough, these abnormal oligosaccharides are still transferred to protein. The mannosamine-induced oligosaccharides were separated into neutral and basic fractions on a cation exchange resin. The neutral oligosaccharides ranged in size from hexose3(GlcNAc)2 to hexose10(GlcNAc)2 with the major species being Man5(GlcNAc)2 to Man7(GlcNAc)2. These oligosaccharides were almost completely susceptible to digestion by alpha-mannosidase and by endoglucosaminidase H. The basic oligosaccharides showed anomolous behavior on the Bio-Gel P-4 columns and appeared to be of small size on the standard columns, ranging from hexose2 to hexose4. However, most of these oligosaccharides were susceptible to digestion by endoglucosaminidase H as well as by alpha-mannosidase, suggesting that they were of different size and structure than would be predicted from the gel filtration patterns. Significantly, when the basic oligosaccharides were subjected to chemical N-acetylation, or when the gel filtration columns were run at high pH rather than at the usual pH of 3.0, the basic oligosaccharides migrated like much larger oligosaccharides. These data provide strong evidence to indicate that some mannosamine can be incorporated into the LLO, and that these mannosamine-containing oligosaccharides exhibit unusual properties. Preliminary studies indicated that Madin-Darby canine kidney cells do incorporate label from [3H]mannosamine into the LLO.
Animals↗
Oligosaccharide structures of human colonic mucin.
Purified human colonic mucin was separated into six distinct components by DEAE-cellulose chromatography, and the structures of oligosaccharide side chains from the three most abundant species were determined. Oligosaccharide side chains were isolated from colonic mucin species III, IV, and V after alkaline borohydride reductive cleavage in the presence of sodium borotritide. After initial separation of acidic and neutral oligosaccharides by ion exchange chromatography, individual oligosaccharides were isolated by sequential chromatography on Bio-Gel P-4 and Bio-Gel P-2 resins followed by preparative normal phase high performance liquid chromatography. Composition and structure of individual oligosaccharides were determined by combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Collectively, 21 discrete oligosaccharide structures were identified in the major human colonic mucin species including 10 acidic oligosaccharides and 11 neutral structures which ranged in size from 2 to 12 sugar residues. Although detailed structures were defined for each oligosaccharide, the majority of the structures identified were variations of a relatively small number of "basic" structures, and several generalizations pertained. First, many oligosaccharides represented variations of a biantennary structure in which branch chains arise in N-acetylglucosaminyl residues linked to C3 and C6 of a galactosyl residue linked in turn to a GlcNAc beta (1-3)GalNAc core; second, non-branched oligosaccharides appeared to be linear chain derivatives of the same core structure; third, all acidic oligosaccharides could be derived from neutral structures present in the mucin species; fourth, sialic acid substitution was limited to few sites and always included substitution in alpha 2-6 linkage to the reducing terminal N-acetylgalactosamine, and finally several structures contained both sialic acid and fucose residues. Individually, mucin species III, IV, and V were found to contain unique mixtures of 13, 14, and 10 oligosaccharide structures, respectively. These data demonstrate that human colonic mucin contain a wide range of oligosaccharides reflecting variations of common core oligosaccharide structures. The major chromatographically defined constituents of normal colonic mucin appear to possess characteristic and distinguishable combinations of oligosaccharide structures. These findings support the concept that colonic mucin contains structurally and functionally distinct subpopulations.
Carbohydrate Conformation↗
Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis.
The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.
Amino Sugars↗
Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins.
Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAE-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and 'H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc alpha 2Ga1 beta 4) or blood group A (Fuc alpha 2(Ga1NAc alpha 3) (Ga1 beta 4) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the beta 3-linked G1cNAc at branch points, whereas the beta 6-linked G1cNAc residue ultimately forms short side chains with a Fuc alpha 2(Ga1NAc alpha 3) Ga1 beta 4 G1cNAc beta 6 structure in individuals with A blood group determinant. The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. [formula: see text] This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of alpha 2-linked fucose to the beta 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.
Amino Acids↗