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PubMed1998

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis.

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.

Carbohydrate Conformation↗
PubMed1999

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.

Base Sequence↗
PubMed2003

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4.

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.

Bacterial Capsules↗
PubMed2003

Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants.

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001). Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios. The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 [SE]) than the remaining infants (8.5 +/- 0.8; p<0.001). Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042). Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea. We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk.

Adult↗
PubMed2004

Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library.

The variation in the sulfation profile of chondroitin sulfate (CS)/dermatan sulfate (DS) chains regulates central nervous system development in vertebrates. Notably, the disulfated disaccharide D-unit, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate), correlates with the promotion of neurite outgrowth through the DSD-1 epitope that is embedded in the CS moiety of the proteoglycan DSD-1-PG/phosphacan. Monoclonal antibody (mAb) 473HD inhibits the DSD-1-dependent neuritogenesis and also recognizes shark cartilage CS-D, which is characterized by the prominent D-unit and is also recognized by two other mAbs, CS-56 and MO-225. We investigate the oligosaccharide epitope structures of these CS-D-reactive mAbs by ELISA and oligosaccharide microarrays using lipid-derivatized CS oligosaccharides. CS-56 and MO-225 recognized the octa- and larger oligosaccharides, though the latter also bound one unique hexasaccharide D-A-D, where A denotes the disaccharide A-unit GlcUA-GalNAc(4-O-sulfate). The octasaccharides reactive with CS-56 and MO-225 shared a core A-D tetrasaccharide, whereas the neighboring structural elements located on the reducing and/or nonreducing sides of the A-D gave a differential preference additionally to the recognition sequence for each antibody. In contrast, 473HD reacted with multiple hexa- and larger oligosaccharides, which also contained A-D or D-A tetrasaccharide sequences. Consistent with the distinct specificity of 473HD as compared with CS-56 and MO-225, the 473HD epitope displayed a different expression pattern in peripheral mouse organs as revealed by immunohistology, extending the previously reported CNS-restricted expression. The epitope of 473HD, but not of CS-56 or MO-225, was eliminated from DSD-1-PG by digestion with chondroitinase B, suggesting the close association of L-iduronic acid with the 473HD epitope. Despite such supplemental information, the integral epitope remains to be isolated for identification and comprehensive analytical characterisation. Thus novel information on the sugar sequences containing the A-D tetrasaccharide core was obtained for the epitopes of these three useful mAbs.

Animals↗
PubMed2005

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii.

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.

Animals↗
PubMed1976

Hydrogen production in the rat following ingestion of raffinose, stachyose and oligosaccharide-free bean residue.

Raffinose and stachyose were compared to cooked California Small White beans (CSW) containing 4% alpha-oligosaccharides (stachyose and raffinose) and to oligosaccharide-free CSW solids (residue from hexane and 70% ethanol extraction of CSW) as sources of hydrogen when ingested by rats maintained in life support systems. If the oligosaccharide content were the only hydrogen source in CSW, it would have had to be 25 times as potent as CSW, but raffinose was only five times and stachyose seven times as potent as CSW. Oligosaccharide-free residue was 0.4 to 0.5 as active as CSW. Hydrogen producing potencies of stachyose and raffinose were enhanced by feeding in combinations with residue. The increases in hydrogen production from the combinations were more than additive. Thus, CSW contains at least one 70% alcohol-insoluble substance which, in addition to the oligosaccharides, is essential to bring about quantitatively the physiological response to whole beans observed in rats.

Animals↗
PubMed2000

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase.

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.

Animals↗
PubMed1987

Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase.

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccharides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4Cer, II3NeuGc-GgOse3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reactivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NH2-HPTLC plate, reductive amination was found to be superior to the heating method reported earlier.

Animals↗
PubMed1992

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

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

Animals↗
PubMed1994

Microbial endoglycosidases for analyses of oligosaccharide chains in glycoproteins.

Microbial endoglycosidases are useful for elucidating the structure and function of the oligosaccharide chains of glycoconjugates. Most of the microbial endo-beta-N-acetylglucosaminidases including Endo-H can preferentially act on high-mannose type chains of asparagine-linked oligosaccharides of various glycoproteins. Among them, Flavobacterium sp. enzyme is produced in large amounts by the inducing cultivation. Using this enzyme, the role of oligosaccharide chains in various microbial glycoenzymes such as Rhizopus glucoamylase, and yeast invertase was examined. The findings suggested that the oligosaccharide chains of them are essential participants in the stabilization of the enzyme and in the protection from proteolytic inactivation. Novel endo-beta-N-acetylglucosaminidases were also found in the culture broths of microorganisms. Unlike most microbial endo-beta-N-acetylglucosaminidases, Endo-M of Mucor hiemalis could act on a complex type oligosaccharide chains, which is similar to Endo-F2 in multiple form of Endo-F from Flavobacterium meningosepticum. The complete amino acid sequences of Endo-F1, -F2, -F3, -H, and Flavobacterium sp. enzyme were determined. All of them had two highly conserved regions common to a number of chitinases. Endo-alpha-N-acetylgalactosaminidase which hydrolyzes the O-glycosidic linkages in glycoproteins was found in the culture broth of only a few microorganisms. The production of Alcaligenes sp. enzyme was highly induced by the addition of porcine gastric mucin in the culture medium. There is some evidence that endo-alpha-N-acetylgalactosaminidases may recognize not only the glycon but also the aglycon amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1979

Analyses of oligosaccharides by tagging the reducing end with a fluorescent compound. I. Application to glycoproteins.

The reducing end sugar of an oligosaccharide and 2-aminopyridine were linked by means of reductive amination with sodium cyanoborohydride. The fluorescent derivative of the oligosaccharide thus obtained, which had a positive charge, was subjected to two-dimensional paper electrophoresis. In the first direction, the sugar derivative moved according to its degree of polymerization, and in the second direction, it moved according to the structure of the borate complex. In this way fluorescent derivatives of saccharides were mapped on a sheet of paper. The method was applied to some known mono- and oligosaccharides and to the saccharides obtained by nitrous deamination of the oligosaccharide portions of glycoproteins (fetuin, Take-amylase A, and ovalbumin). The fingerprints thus obtained were characteristic of the chemical structures of the original oligosaccharides.

Aminopyridines↗
PubMed1980

The asparagine-linked sugar chains of the glycoproteins in calf thymocyte plasma membrane. Structural studies of neutral oligosaccharides.

The neutral oligosaccharide fraction obtained from the hydrazinolysate of the plasma membrane glycoproteins of calf thymocytes was shown to be a mixture of twelve oligosaccharides. Oligosaccharides A, B, and C, which released galactose on treatment with jack bean beta-galactosidase but not with diplococcal beta-galactosidase, were shown to have the structures Gal beta 1-3Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-6(3)[Gal beta 1-3Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3(6)]Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc, Gal beta 1-3Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-6(3)[Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3(6)]Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc, and Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc, respectively. Oligo saccharides D and E, which released galactose on treatment with either jack bean beta-galactosidase or diplococcal beta-galactosidase, were shown to have the structures Gal beta 1-4GlcNAc beta 1-2Man alpha 1-(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-)GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-)GlcNAc, respectively. Five oligosaccharides (F1, G, H1, I, and J) were shown to have typical high-mannose type structures (Man alpha 1-)n.Man beta 1-4GlcNAc beta 1-GlcNAc, with n-values of 8, 7, 6, 5, and 4, respectively. The structures of the remaining two oligosaccharides F2 and H2 were elucidated as GlcNAc beta 1-2Man alpha 1-(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-)GlcNAc and Man alpha 1-(Man alpha 1-)Man beta 1-(GlcNAc beta 1-Man alpha 1-)Man beta 1-GlcNAc beta 1-GlcNAc, respectively.

Animals↗
PubMed1985

Neutral oligosaccharides in the urine of a patient with glycogen storage disease type II.

Neutral oligosaccharides were isolated from urine of an adult patient with glycogen storage disease type II, a deficiency of lysosomal acid alpha-glucosidase, by chromatography on columns of activated charcoal, Dowex 50 X 2 and Dowex 1 X 2. Total neutral oligosaccharides in the urine of the patient were increased about 5-fold as compared with those in normal controls. The most accumulated oligosaccharide was separated by Bio-Gel P-2 column chromatography, and finally purified by paper chromatography. Based on various studies, including carbohydrate analysis, chemical ionization mass spectrometry, fast atom bombardment mass spectrometry, degradation by glucoamylase and isopullulanase, and methylation analysis, the structure of this oligosaccharide was deduced to be Glc alpha 1----6Glc alpha 1----4Glc alpha 1----4Glc. This oligosaccharide appears to be accumulated in urine of the patient with acid alpha-glucosidase deficiency as an end product of the hydrolysis of glycogen.

Carbohydrate Sequence↗
PubMed1986

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts.

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.

Amino Acids↗
PubMed1990

Analysis of the oligosaccharides on rat androgen-binding protein using serial lectin chromatography.

The microheterogeneity seen when rat androgen-binding protein (rABP) is analyzed by two-dimensional polyacrylamide gel electrophoresis is attributable, at least in part, to the differential glycosylation of a single promoter. Further insight into the chemical nature of the oligosaccharide units on rABP was obtained by serial lectin chromatography. When rABP was chromatographed on immobilized Concanavalin A (Con-A), it was fractionated into three classes: (1) one that did not bind to the lectin (about 44% of the rABP), (2) one that was bound and could be eluted with 10 mM 1-O-methyl alpha-D-glucopyranoside (glucoside), about 34%, and (3) one that could be eluted with 0.5 M methyl alpha-D-mannopyranoside (mannoside), about 23%. Binding to Con-A indicates the presence of asparagine-linked oligosaccharides. Chromatography of the glucoside-eluted peak on lentil lectin (LcH) indicated that the rABP in that fraction contained a fucose residue on the chitobiose core. Chromatography of the mannoside-eluted peak on wheat germ agglutinin (WGA) indicated the presence of rABP with high mannose- (44%) and hybrid-type (56%) glycans attached. Chromatography on Ricinus communis I (RCA-I) lectin indicated a species containing galactosylated complex-type oligosaccharide chains. Treatment of rABP forms with exoglycosidases confirmed the presence of externally disposed fucose, sialic acid, mannose, and galactose residues. LcH chromatography indicated that about 30% of the rABP that did not bind to Con-A possessed triantennary oligosaccharides with fucose on the chitobiose core. About 28% of the rABP was retarded when it was chromatographed on Phaseolus vulgaris E lectin, suggesting the presence of bisected biantennary chains with terminal galactose residues. We were unable to detect rABP species with serine- or threonine-linked oligosaccharide chains in this fraction. Other forms of rABP in the nonretained fraction of Con-A were not resolved. Western blotting did not reveal major differences in relative molecular weight (Mr) among the rABP species; some differences in the ratio of the heavy to the light subunit of the molecule were detectable.

Androgen-Binding Protein↗
PubMed2001

Equine follicle-stimulating hormone: molecular cloning of beta subunit and biological role of the asparagine-linked oligosaccharide at asparagine(56) of alpha subunit.

Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated alpha and beta subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn(56) of the alpha subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH beta subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH alpha56/beta) to investigate the biological role of the oligosaccharide at Asn(56) in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG alpha56/beta) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH alpha56/beta), however, did not show any FSH activity, indicating that the oligosaccharide at Asn(56) was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn(56) of the alpha subunit.

Amino Acid Sequence↗
PubMed2000

Variability of human milk neutral oligosaccharides in a diverse population.

BACKGROUND: A complex array of free oligosaccharides is a distinctive compositional feature of human milk. Although these oligosaccharides have been studied for several years, their variability and distribution have not been systematically studied, and their nutritional and functional roles have not been elucidated. This report describes a study in which a large number of human milk samples were analyzed for the presence and content of nine neutral oligosaccharides. The resultant data were used to probe for distribution trends by donor groups and stage of lactation. METHODS: Milk samples from 435 women residing in 10 countries were analyzed using a simple preparation procedure, gel filtration, and high-performance anion-exchange chromatography. RESULTS: All samples contained structures based on lacto-N-neotetraose and lacto-N-tetraose. This contrasts with the fucosyloligosaccharides tested, none of which was detected in 100% of the samples. Unexpected distribution trends were observed. For example, 100% of the samples from Mexico (n = 156) contained 2'-fucosyllactose, whereas only 46% of the samples from the Philippines (n = 22) contained this structure. Concentration ranges for the analyzed oligosaccharides revealed quantitative and qualitative distribution trends. CONCLUSIONS: The oligosaccharide composition of human milk varied among samples. The geographical origin of the donors was one of the factors that accounted for this variability. This can be explained by genetically determined traits that are not uniformly distributed. Results indicated that further systematic studies are needed to ascertain the effect of other factors, such as lactation stage or diet.

Asia↗