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.     

An investigation of the interactions of E-selectin with fuco-oligosaccharides of the blood group family

This investigation is concerned with assignments of Lewis(a) (Le(a)) and Le(x) analogs on linear and branched di- to hexasaccharide backbones as components of the recognition motifs for E-selectin. The influence of the location of fucose residue(s) was investigated using 14 structurally defined and variously fucosylated oligosaccharides in biotinylated form or as neoglycolipids in static binding assays, in microwells, and on thin-layer chromatograms. Results of the two assay systems were in agreement overall and showed that the recognition motifs for E-selectin include 4-fucosyl-lacto (Le(a)) and 3-fucosyl-neo-lacto (Le(x)) sequences strictly at capping positions and not Le(x) at an internal position as a part of VIM-2 antigen sequence. There is greater potency of the Le(a) over the Le(x) series. Additional fucose residues alpha1-2-linked to neighboring galactoses or alpha1-3-linked to inner N-acetyglucosamines or to reducing-terminal glucose residues of the tetrasaccharide backbone had little or no effect on the selectin binding. E-selectin binding to the Le(a) or Le(x )capping motif on a 3-linked branch was equivalent to the binding on the corresponding linear backbone. A lack of E-selectin binding to the Le(x) motif capping a 6-linked branch and to the Le(x) trisaccharide linked to biotin via a nine-carbon spacer indicates that the -GlcNAcbeta1-3Gal- sequence on the oligosaccharide backbone adjoining the Le(x) is a part of recognition motif for E-selectin. These findings contribute to understanding the molecular basis of E-selectin recognition and could influence future designs of selectin antagonists as possible therapeutic substances.     

Structures of neutral oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Le(a+b-)) patient suffering from chronic bronchitis.

Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitis patient with blood group O, Le(a+b-). Oligosaccharides were released by alkaline-borohydride treatment and purified by anion-exchange chromatography, size exclusion chromatography, and HPLC on a silica-bonded alkylamine column. Structural studies employed 500-MHz 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry and methylation analysis. Twenty-four neutral oligosaccharides, ranging in size from disaccharides to hexasaccharides, were fully characterized in this study. None of the structures contained an alpha(1----2)-linked fucose residue, in keeping with the non-secretor status of the patient. Seven of the structures had fucose present in alpha(1----3)-linkage in the X-determinant, while only one oligosaccharide (compound 14b) was seen with fucose alpha(1----4)-linked in the Le(a) determinant. The following eight structures isolated from the mucins of the non-secretor patient had not been found previously in the mucins of secretor individuals: [formula: see text] This study confirms that the blood group status of an individual is reflected in the carbohydrate structures of the secreted mucins. Furthermore, it clearly illustrates the need for detailed carbohydrate structural studies of mucins from different individuals before any attempt can be made to correlate observed differences in oligosaccharide profiles to disease status.     

Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection

The most common cause of infant mortality is diarrhea; the most common cause of bacterial diarrhea is Campylobacter jejuni, which is also the primary cause of motor neuron paralysis. The first step in campylobacter pathogenesis is adherence to intestinal mucosa. We found that such binding was inhibited in vitro by human milk and, with high avidity, by alpha1,2-fucosylated carbohydrate moieties containing the H(O) blood group epitope (Fuc alpha 1,2Gal beta 1,4GlcNAc em leader ). In studies on the mechanism of adherence, campylobacter, which normally does not bind to Chinese hamster ovary cells, bound avidly when the cells were transfected with a human alpha1,2-fucosyltransferase gene that caused overexpression of H-2 antigen; binding was specifically inhibited by H-2 ligands (lectins Ulex europaeus and Lotus tetragonolobus and H-2 monoclonal antibody), H-2 mimetics, and human milk oligosaccharides. Human milk oligosaccharides inhibited campylobacter colonization of mice in vivo and human intestinal mucosa ex vivo. Campylobacter colonization of nursing mouse pups was inhibited if their dams had been transfected with a human alpha1,2-fucosyltransferase gene that caused expression of H(O) antigen in milk. We conclude that campylobacter binding to intestinal H-2 antigen is essential for infection. Milk fucosyloligosaccharides and specific fucosyl alpha1,2-linked molecules inhibit this binding and may represent a novel class of antimicrobial agents.     

Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.     

A novel sialylfucopentaose in human milk. Presence of this oligosaccharide is not dependent on expression of the secretor or Lewis fucosyltransferases

We have identified a novel oligosaccharide in human milk that is a fucosyl derivative of sialyltetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc). This oligosaccharide was purified by affinity chromatography on a column of immobilized Ricinus communis I lectin. Structural analyses of radiolabeled oligosaccharides by exoglycosidase digestions, binding by specific anti-carbohydrate antibodies, and analysis of the 3H-labeled glucitol derivative obtained after permethylation and hydrolysis are consistent with the following proposed structure. (formula; see text) The analyses of human milk sialylpentasaccharides from donors typed as Le(a-,b+), Le(a+,b-), and Le(a-,b-) secretor confirmed the secretor gene-dependent expression of the sialylated lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and the Lewis gene-dependent expression of the sialylated lacto-N-fucopentaose II (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-4Glc). However, the presence of this novel oligosaccharide in human milk is not dependent on the expression of either the secretor gene or the Lewis gene-specified fucosyltransferases.     

Structural characterization of the oligosaccharide chains of native and crystallized boar seminal plasma spermadhesin PSP-I and PSP-II glycoforms.

The PSP-I/PSP-II heterodimer is the major protein of boar seminal plasma. Both subunits are glycoproteins of the spermadhesin family and each contains a single N-glycosylation site. After enzymatic release of the oligosaccharides from isolated PSP-I and PSP-II, mainly neutral and monosialylated oligosaccharides, and small amounts of disialylated oligosaccharides, were recovered from both proteins. Twenty-two neutral oligosaccharides, 11 monosialylated glycans and three disialylated carbohydrate chains were characterized using mass spectrometric and NMR techniques. PSP-I and PSP-II share the same glycans but differ in their relative molar ratios. Most glycan structures are proximally alpha1-6-fucosylated, diantennary complex-type bearing nonsialylated or alpha2-6-sialylated N-acetyllactosamine or di-N-acetyllactosamine antennae. The majority of nonsialylated N-acetyllactosamine antennae bear terminal alpha1-3-linked Gal residues. In addition, the N-acetylglucosamine residue of nonsialylated N-acetyl and di-N-acetyllactosamine antennae can be modified by an alpha1-3-linked fucose residue. Structures of higher antennarity, as well as structures 3,6-branched at galactose residues, were found in smaller amounts. In one oligosaccharide, N-acetylneuraminic acid is substituted by N-glycolylneuraminic acid. Mass spectrometric analysis of PSP-I and PSP-II glycoforms isolated from crystallized PSP-I/PSP-II heterodimer showed the coexistence of major PSP-I and PSP-II glycoforms in the hexagonal crystals. Oligosaccharides with the NeuNAcalpha2-6GalNAcbeta1-4GlcNAc-R motif block adhesive and activation-related events mediated by CD22, suggesting a possible immunoregulatory activity for PSP-I/PSP-II.     

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.     

Primary structure of the glycans from mouse serum and milk transferrins.

A 'serotransferrin-like' protein was purified from mouse milk. This serotransferrin cross-reacts immunologically with the serotransferrin isolated from mouse plasma and not with the mouse lactotransferrin (lactoferrin). Sugar analysis of the three transferrins, i.e. serotransferrin, milk 'serotransferrin-like' protein and lactotransferrin, revealed that the major difference between the glycan primary structure of mouse serotransferrin and those of mouse milk 'serotransferrin-like' protein and lactotransferrin concerns essentially the presence of one fucose residue in the last two proteins. For structural determination, the N-glycosidically linked glycans were released from the protein by a reductive cleavage of the oligosaccharide-protein linkage under strong alkaline conditions. The primary structure of the released oligosaccharide alditols was determined by methylation analysis and 400 MHz 1H-n.m.r. spectroscopy. The oligosaccharide alditols released from milk 'serotransferrin-like' protein and lactotransferrin were identical and were identified as disialylated biantennary glycans of the N-acetyl-lactosamine type with a fucose residue alpha-1,6-linked to the N-acetylglucosamine residue conjugated to the peptide chain and having the following primary structure: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-N)Asn. The serotransferrin glycan has the same primary structure but is only partially fucosylated (10-15%).     

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

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

Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding. E-selectin binding is unaffected in the presence of the blood group H fucose (alpha 1-2 linked to galactose to form the Le(b) antigen). However, the binding is abolished when in addition alpha 1-3-linked N-acetylgalactosamine to the galactose (blood group A antigen) is present. These results indicate that some E-selectin-mediated adhesive events may be influenced by blood group status.     

Identification and characterization of glycosylation sites in human serum clusterin.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.     

Cooperative Interactions of Oligosaccharide and Peptide Moieties of a Glycopeptide Derived from IgE with Galectin-9

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo- and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.     

Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0. 2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo (31)P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha1-6-linked fucose were detected that carried no, one or two beta1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcalpha2-3Galbeta1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed.     

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.     

Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides were GalGlcNAcMan(6)GlcNAc-Fuc(0-1)GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man(5-7)GlcNAc(2) were the predominant species (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.     

Increased sialylation and defucosylation of plasma proteins are early events in the acute phase response

Within hours of turpentine injection to stimulate the acute phase (AP) response in rats, the N-acetylneuraminic acid content of plasma proteins increases and that of fucose decreases, each by about 60%. The two changes are inversely related (r = -0.97). The NeuAc/Gal ratio increases from the normal 0.75 to 1.0 on day 2 of the AP. Whereas 50% of the isolated oligosaccharides of normal plasma proteins are retarded on immobilized Ricinus communis agglutinin, those from day 2 AP plasma fail to do so. This indicates that NeuAc caps the normally Gal-terminated chains. alpha1-Acid glycoprotein (a positive AP protein), alpha1-macroglobulin (a non-AP protein), and alpha1-inhibitor3 (a negative AP protein) also show similar alterations in NeuAc/Gal ratio and decreases in Fuc. alpha2-Macroglobulin, which arises only during the AP, does not contain significant amounts of Fuc. Sambucus nigra agglutinin (alpha2,6-linked NeuAc-specific) binds a majority of plasma proteins, and binding is increased during the AP response. Maackia amurensis lectin (alpha2,3-linked NeuAc-specific) binds only three proteins in normal plasma and three additional proteins in AP plasma. The Fuc-specific Aleuria aurantia agglutinin and Lens culinaris agglutinin each detect five proteins in normal plasma. Their binding decreases during the AP response. These results show that: (1) sialylation and defucosylation of preexisting plasma proteins occur rapidly in the AP response; (2) sialylation caps the preexisting Gal-terminating oligosaccharides; and (3) the oligosaccharides of even the non-AP and negative AP proteins are modified. These changes are distinct from the elevation in the levels of protein-bound monosaccharides and the altered concanavalin A-binding profile the oligosaccharides of AP proteins acquire in diseases.     

Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha

Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.     

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.      

The structure of carbohydrate unit B of porcine thyroglobulin.

The oligosaccharide fraction was obtained from porcine thyroglobulin by hydrazinolysis. Four fractions of unit B-type oligosaccharides were purified by successive chromatographies on columns of DEAE-cellulose and concanavalin A-Sepharose, and their structures were investigated by the combination of endo- and exo-glycosidase digestions, methylation analysis and Smith degradation. From the results of these studies, the structures of the unit B oligosaccharides were proposed to be as follows: see formula in text. Thus the glycoprotein was found to have triantennary and biantennary complex-type oligosaccharides as acidic sugar chains. Concerning the triantennary oligosaccharides, the following structural features were shown: (1) the sialic acid residues were not localized on certain specific branches but distributed on all three branches; (2) however, alpha (2 leads to 3)-linked sialic acid residues were exclusively located on the terminal of the branch arising from C-4 of the branching alpha-mannose residue, whereas alpha (2 leads to 6)-linked sialic acid residues occupied terminals of the other branches; (3) the outer branching alpha-mannose residue was attached to C-3 or C-6 of an inner branching beta-linked mannose residue, and both types were observed to exist.