The structures of the asparagine-linked sugar chains of human apolipoprotein B-100

The asparagine-linked sugar chains of human apolipoprotein B-100 were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Their structures were elucidated by sequential exoglycosidase digestion in combination with methylation analysis after fractionation by paper electrophoresis and gel permeation chromatography. One neutral and two acidic fractions were obtained by paper electrophoresis in a molar ratio of 7:8:5. The neutral fraction contained high-mannose type oligosaccharides consisting of Man5GlcNAc2 to Man9GlcNAc2. The acidic fractions contained monosialylated and disialylated biantennary complex type oligosaccharides. As minor components in the monosialylated fraction, biantennary complex-type oligosaccharides which were absent one terminal galactose residue, monoantennary complex type, and hybrid type oligosaccharides were detected. Apolipoprotein B-100 was calculated to contain 5-6 mol of high-mannose type and 8-10 mol of complex type oligosaccharides per mole protein.     

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le^a and A, Le^b). Neutral and acidic components were separated from each other by anion-ecchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore, the molar response factors after HPAEC-PAD have been determined for 8 components.     

Incorporation of orally applied (13)C-galactose into milk lactose and oligosaccharides

Biosynthesis and functions of human milk oligosaccharides (HMO) are not well known. A typical housekeeping enzyme, beta1,4-galactosyltransferase, links galactose to glucose to form lactose which is then used as backbone for the assembly of HMO. We investigated whether milk lactose and HMO may be labeled in vivo by an orally given (13)C-galactose bolus. Eleven exclusively breastfeeding mothers were given a (13)C-galactose bolus at the end of their breakfast. Milk and urine samples from each nursing up to 36 h were analyzed for carbohydrate composition by high-performance thin-layer chromatography, high-pH anion-exchange chromatography, and fast atom bombardment mass spectrometry. (13)C enrichment of milk fractions, urinary carbohydrates, lactose, and oligosaccharides as well as of breath CO(2) was determined by isotope ratio mass spectrometry. Up to 10% of the orally given galactose bolus was directly transported to the mammary gland and incorporated into milk components. Characteristic for most milk samples was the appearance of two (13)C-peaks, the first immediately after the (13)C-bolus was taken and the second on the next morning. The highest (13)C enrichment was found in lactose followed by neutral and acidic oligosaccharides. In breath samples, the (13)C-excretion followed the same pattern as in milk. (13)C nuclear magnetic resonance of isolated lactose revealed (13)C only at C(1)-atom of galactose and C(1)-atom of glucose. This label was without any exception at the same position as the (13)C-label of the orally applied galactose. Neutral and acidic HMO can easily be (13)C-labeled in vivo which facilitates investigations of their metabolic fate in infants.     

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.     

Quantitative analysis of oligosaccharide structure of glycoproteins

A sensitive and quantitative method for the structural analysis of oligosaccharide was established for the glycoform analysis of glycoproteins. In this study,N-linked oligosaccharides of human IgG and bovine transferrin were analyzed for the evaluation of the method. Carbohydrate moiety of glycoprotein was released by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled with a fluorescent dye, 2-aminobenzamide, for the enhancement of detection sensitivity. Sialylated (acidic) oligosaccharides were separated from neutral oligosaccharide by employing a strong anion-exchange column (MonoO) followed by the treatment with sialidase. Enzymatically desialyated fractions and neutral fractions of oligosaccharides were applied to normal-phase HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential digestion with exoglycosidases. As a result, disialylated biantennary complextype oligo saccharide was found to be a major sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant. These results coincided well with reported results.     

Structural analysis of five new monosialylated oligosaccharides from human milk.

The total monosialylated oligosaccharide fraction from pooled human milk was isolated by gel filtration and ion-exchange chromatography. Further separation by HPLC using a mobile phase containing an ion-pairing reagent of triethylamine gave five new monosialylated oligosaccharides. Structural analysis was carried out by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz NMR spectroscopy. Combined structural data revealed the following new structures: [formula: see text]     

Carbohydrate Structures of β-Trace Protein from Human Cerebrospinal Fluid: Evidence for "Brain-Type"N-Glycosylation

Abstract: The carbohydrate structures of β-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn²⁹ and Asn⁵⁶). Enzymatically released N -glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N -acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N -acetylglucosamine residues of nearly all structures were found to be α1,6-fucosylated. Peripheral fucose (about 20%α1,3-linked to N -acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N -acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N -acetylglucosamine only were present. At least 20 different N -glycans were identified. Analysis of the site-specific glycosylation patterns at Asn²⁹ and Asn⁵⁶ revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N -acetylglucosamine, as well as terminal N -acetylglucosamine and galactose residues, and significant amounts of N -acetylneuraminic acid in α2,3 linkage), this protein can be classified as "brain-type" glycosylated.     

A detailed analysis of neutral and acidic carbohydrates in human milk.

Reverse- and normal-phase chromatography have been used to separate a number of standard human milk oligosaccharides derivatized via a reductive amination reaction with 2-aminoacridone (2-AMAC). Analytes were detected by spectrofluorimetry and injected simultaneously with a hydrolyzed dextran ladder derivatized with methyl-4-aminobenzoate. The latter probe does not fluoresce at the wavelength of emission by the 2-AMAC derivatives, and the derivatized, hydrolyzed dextran components were visualized by their ultraviolet absorbance. This procedure gave precise measurements of the "size" of 2-AMAC oligosaccharides in terms of their glucose equivalent values. Analytical amounts of 2-AMAC oligosaccharide standards were also isolated for further characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. MS-MS was also used to provide information on oligosaccharide sequences. This methodology was used successfully to characterize mixtures of neutral and acidic oligosaccharides from samples of human milk. This approach could be usefully applied to the study of glycoforms from a variety of samples such as those released from glycoproteins/glycolipids; these have been reported to be altered in a number of diseases, for example, cancer, cystic fibrosis, and autoimmune disease such as rheumatoid arthritis.     

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

Oligosaccharides from feces of preterm infants fed on breast milk

Nine neutral and five acidic oligosaccharides were isolated from feces of a preterm (30th postmenstrual week) blood group A nonsecretor infant fed on pooled breast milk. Structural analyses were carried out using sugar and methylation analyses, fast atom bombardment mass spectrometry, and 1H NMR. The acidic oligosaccharides are well-known components of human milk. The neutral oligosaccharides are characteristic of nonsecretor milk. Surprisingly, no secretor gene-dependent oligosaccharides were present in the feces. Another preterm (27th postmenstrual week) blood group A, secretor infant fed on pooled breast milk showed the same fecal oligosaccharide pattern as above during the first week after birth, despite being a secretor individual. Also notable was the absence of blood group A-active oligosaccharides in this sample. Another sample of feces collected 8 weeks later from the latter infant contained the expected blood group A-active oligosaccharides. Furthermore, free sialic acid was present at the cost of the sialyl oligosaccharides seen earlier. Thus, infants born prematurely do not show the same degree of development of oligosaccharide metabolism as their more mature counterparts.     

Characterization of oligosaccharides in milk of bearded seal (Erignathus barbatus)

Carbohydrates were extracted from milk of a bearded seal, Erignathus barbatus (Family Phocidae). Free neutral oligosaccharides were separated by gel filtration, anion-exchange chromatography and preparative thin layer chromatography, while free acidic oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows; lactose, 2'-fucosyllactose, lacto-N-fucopentaose IV, difucosyl lacto-N-neohexaose and difucosyl decasaccharide which contained a lacto-N-neohexaose unit as well as an additional Gal(beta1-4)GlcNAc(beta1-3) unit and two residues of non-reducing Fuc(alpha1-2). The acidic oligosaccharides were thought to contain an Neu5Ac(alpha2-6) residue linked to GlcNAc or a sulfate linked to Gal at OH-3. The sialyl oligosaccharides and sulfated oligosaccharides had a lacto-N-neohexaose unit and two non-reducing Fuc(alpha1-2) residues and some of them had in addition one or two Gal(beta1-4)GlcNAc(beta1-3) units. The milk oligosaccharides of the bearded seal were compared to those of the harbour seal, which had been studied previously.     

Fractionation of sialyl oligosaccharides of human milk by ion-exchange chromatography

The sialyl oligosaccharides of human milk are partially separated by ion-exchange chromatography on DEAE-cellulose in acetic acid-pyridine buffers, pH 5.4. One neutral and seven sialyl oligosaccharide fractions are obtained in essentially quantitative yields. The method permits the large-scale preparation of sialyl oligosaccharide fractions from which individual sialyl oligosaccharides may be purified without the necessity of preparative high-voltage paper electrophoresis.     

The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides

The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.     

The Asn-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male. Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains.

Human Tamm-Horsfall glycoprotein has been purified from the urine of one male. The Asn-linked carbohydrate chains were enzymically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, and separated from the remaining protein by gel-permeation chromatography on Bio-Gel P-100. Fractionation of the intact (sulfated) sialylated carbohydrate chains was achieved by a combination of three liquid-chromatographic techniques, namely, anion-exchange FPLC on Q-Sepharose, amine-adsorption HPLC on Lichrospher-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. In total, more than 150 carbohydrate-containing fractions were obtained, some of which still contained mixtures of oligosaccharides. The primary structure of 30 N-glycans, including 10 novel oligosaccharides, were determined by one- and two-dimensional 1H-NMR spectroscopy at 500 MHz or 600 MHz. The types of compounds identified range from non-fucosylated, monosialylated, diantennary to fucosylated, tetrasialylated, tetraantennary carbohydrate chains, possessing the following terminal structural elements: [formula: see text]     

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.     

Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein

Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.     

Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.