Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection

Townsend, R. Reid; Hardy, Mark R.; Cumming, Dale A.; Carver, Jeremy P.; Bendiak, Brad

doi:10.1016/0003-2697(89)90708-2

Cited by 165 publications

(77 citation statements)

References 13 publications

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“…We characterized the gliding on uniform oligosaccharides in detail and compared it with the gliding on the plastic array and glass covered with mixed SOs. The plastic array and glass covered by mixed SOs were prepared by using (i) serum containing various sialylated glycoconjugates (32), (ii) fetuin (33,34), a serum protein that attaches three Olinked oligosaccharide molecules, and three N-linked oligosaccharide molecules derived from 23 known structures, and (iii) a mixture of serum and fetuin (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Gliding Motility of Mycoplasma mobile on Uniform Oligosaccharides

2015

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The binding and gliding of Mycoplasma mobile on a plastic plate covered by 53 uniform oligosaccharides were analyzed. Mycoplasmas bound to and glided on only 21 of the fixed sialylated oligosaccharides (SOs), showing that sialic acid is essential as the binding target. The affinities were mostly consistent with our previous results on the inhibitory effects of free SOs and suggested that M. mobile recognizes SOs from the nonreducing end with four continuous sites as follows. (i and ii) A sialic acid at the nonreducing end is tightly recognized by tandemly connected two sites. (iii) The third site is recognized by a loose groove that may be affected by branches. (iv) The fourth site is recognized by a large groove that may be enhanced by branches, especially those with a negative charge. The cells glided on uniform SOs in manners apparently similar to those of the gliding on mixed SOs. The gliding speed was related inversely to the mycoplasma's affinity for SO, suggesting that the detaching step may be one of the speed determinants. The cells glided faster and with smaller fluctuations on the uniform SOs than on the mixtures, suggesting that the drag caused by the variation in SOs influences gliding behaviors. IMPORTANCEMycoplasma is a group of bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide in the direction of the protrusion. These procedures are essential for parasitism. Usually, mycoplasmas glide on mixed sialylated oligosaccharides (SOs) derived from glycoprotein and glycolipid. Since gliding motility on uniform oligosaccharides has never been observed, this study gives critical information about recognition and interaction between receptors and SOs. M ycoplasmas are parasitic and occasionally commensal bacteria that have small genomes and lack a peptidoglycan layer (1). Several Mycoplasma species form membrane protrusions, such as the headlike structure in Mycoplasma mobile (2) and the attachment organelle in the human pathogen Mycoplasma pneumoniae (3-5). On solid surfaces, these species exhibit gliding motility in the direction of the protrusion; this motility appears to be involved in the parasitism of mycoplasmas. Interestingly, mycoplasmas have no flagella or pili, and their genomes contain no genes related to known bacterial motility. In addition, no homologs of motor proteins that are common in eukaryotic motility have been found.M. mobile, isolated from the gills of freshwater fish, is a fastgliding mycoplasma. It glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 m/s (6, 7), or three to seven times the length of the cell per second, exerting a force of up to 27 pN (8-10). The gliding machinery formed at the base of the membrane protrusion can be divided into internal and surface structures. The internal, "jellyfish" structure is composed of about 10 proteins (11, 12). The surface structure is composed of hundreds of units, with a flexible leg protruding from each (13-15). The l...

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Section: Resultsmentioning

confidence: 99%

Gliding Motility of Mycoplasma mobile on Uniform Oligosaccharides

2015

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“…Native acidic oligosaccharides of milk have been resolved into monosialyl-, disialyl-, and trisialyloligosaccharide mixtures by mediumpressure anion-exchange chromatography with absorption at 214 nm [22], but further resolution was not achieved. A technique more commonly employed for native milk oligosaccharide resolution and quantification is high pH anion exchange chromatography with pulsed amperometric detection [17,[23][24][25][26], but the electrochemical response factors for anionic sialyloligosaccharides by this technique are not consistent, and differ by the linkage position of the sialic acid [23]. Reverse-or normal-phase chromatography was able to resolve derivatized or reduced acidic oligosaccharides, with detection by UV absorbance or spectrofluorimetry as the basis for quantification [27,28].…”

Section: Introductory Statementmentioning

confidence: 99%

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

Bao

Zhu

Newburg

2007

Analytical Biochemistry

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The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange HPLC, reverse or normal phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection, and, in our laboratory, by CE with detection at 205 nm. The novel method described herein uses a running buffer of aqueous 200 mM NaH 2 PO 4 at pH 7.05 containing 100 mM SDS made 45% (v/v) with methanol to baseline resolve five oligosaccharides, and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-minute run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants. Keywordshuman milk oligosaccharides; sialyloligosaccharides; capillary electrophoresis INTRODUCTORY STATEMENTHuman milk contains a large number of compounds that provide nutritional support and contribute toward defense of the newborn. Human milk oligosaccharides (HMOS), a principal component of human milk, are found in concentrations of 6 to 12 g/L, and these oligosaccharides vary in size, structure, and abundance [1]. The oligosaccharides of milks from other species vary widely, and the composition of oligosaccharides from human milk is unique [2].HMOS play a potentially protective role to nursing infants. Breastfed infants have a different microbiota than infants fed artificially, and human milk glycans are thought to act as prebiotics that promote the growth of Bifidobacterium bifidus [3]. Breast-fed infants have fewer or less severe gastrointestinal and respiratory infections during the first year of life than formula-fed

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“…4. Previously, the components and structures of N-glycans from bovine fetuin have been well characterized by chromatographic separation, HPAEC-PAD analysis, and detailed NMR studies (67)(68)(69)(70). The majority (Ͼ80%) of the N-glycans were triantennary N-glycans with varied degrees of ␣2,6-and/or ␣2,3-sialylations at the terminus.…”

Section: Generation and Characterization Of Glycosynthase Mutants Of mentioning

confidence: 99%

“…5B). The assignment of the peaks was based on a detailed HPAEC-PAD analysis of fetuin N-glycans reported previously (67,69 (Fig. 5C).…”

Section: Generation and Characterization Of Glycosynthase Mutants Of mentioning

confidence: 99%

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Giddens

Lomino

Amin

et al. 2016

Journal of Biological Chemistry

115

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Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptor substrate specificity toward ␣1,6-fucosyl-GlcNAc-Asn or ␣1, 6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi-and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.Protein glycosylation is one of the most prevalent posttranslational modifications, found in almost all living organisms ranging from bacteria to eukaryotes (1). Glycosylation can profoundly affect a protein's intrinsic properties, such as folding, stability, intracellular trafficking, and immunogenicity. In addition, the oligosaccharide components of glycoproteins can participate directly in a number of important biological recognition processes, including cell adhesion, signaling, hostpathogen interactions, and immune responses (2-8). It is well documented that subtle changes in glycosylation can lead to a significant impact on the biological functions and, in the case of therapeutic glycoproteins, such as monoclonal antibodies, the in vivo stability and therapeutic efficacy (7, 9 -12). Natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ only in the structures of pendant glycans, from which pure glycoforms are extremely difficult to isolate by current chromatographic techniques. As a result, synthetic homogeneous glycopeptides and glycoproteins emerge as indispensable tools for functional studies and for drug/vaccine discoveries. Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemic...

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Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection

Cited by 165 publications

References 13 publications

Gliding Motility of Mycoplasma mobile on Uniform Oligosaccharides

Gliding Motility of Mycoplasma mobile on Uniform Oligosaccharides

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

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