Automated simultaneous release of intact and unreduced N and O-linked glycans from glycoproteins

Merry, Anthony H.; Bruce, Jim; Bigge, Collette; Ioannides, Antonis

doi:10.1042/bst020091s

Cited by 13 publications

(5 citation statements)

References 6 publications

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“…CD164 recombinant proteins were subjected to automated hydrazinolysis (33) using a GlycoPrep 1000 instrument (Oxford GlycoSciences). Released glycans were fluorescently labelled with 2‐aminobenzamide (2AB) by reductive amination according to the method of Bigge et al (34) using an Oxford GlycoSciences Signal TM labelling kit (Oxford GlycoSciences).…”

Section: Methodsmentioning

confidence: 99%

Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis

et al. 2005

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Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

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Section: Methodsmentioning

confidence: 99%

Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis

et al. 2005

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“…Glycosyl Linkage Analysis. The oligosaccharide was released from 28 nmol of the 57-65 glycopeptide by automated hydrazinolysis (Merry et al, 1992) on a GlycoPrep 1000 instrument (Oxford GlycoSystems) and was prereduced with NaBH4. The glycosyl linkage composition of the factor IX oligosaccharide was determined by methylation analysis (Lindberg, 1972) using the procedure of Waeghe etal.…”

Section: Methodsmentioning

confidence: 99%

Identification and structural analysis of the tetrasaccharide NeuAc.alpha.(2.fwdarw.6)Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.fwdarw.3)Fuc.alpha.1.fwdarw.O-linked to serine 61 of human factor IX

Harris¹,

Halbeek²,

Glushka³

et al. 1993

Biochemistry

104

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O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.

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“…In addition, the use of sodium acetate, pH 8.0, in place of sodium bicarbonate increased the buffering capacity of the reaction. These reaction conditions are similar to those developed for automated hydrazinolysis (2,25,26).…”

Section: Modifications To Sample Preparation and Glycan Recovery Procmentioning

confidence: 95%

“…Therefore, a more appropriate technique was developed. Glass beads have been employed in automated hydrazinolysis as an alternative matrix for adsorption of glycans (2,25,26) for sample cleanup following the hydrazinolysis reaction and this procedure was adapted for the optimized manual glycan processing procedure. Following hydrazine evaporation and glycan re-N-acetylation, an aqueous solution of glycans was absorbed onto the glass beads, as described under Materials and Methods.…”

Section: Modifications To Sample Preparation and Glycan Recovery Procmentioning

confidence: 99%

Recovery of Intact 2-Aminobenzamide-Labeled O-Glycans Released from Glycoproteins by Hydrazinolysis

Merry

Neville

Royle

et al. 2002

Analytical Biochemistry

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115

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Automated simultaneous release of intact and unreduced N and O-linked glycans from glycoproteins

Abstract: Oligosaccharidesingl co roteinsmay generally be attached to eptides in two ways, gy 8-glycosidic linkage to asparagine or g 0-glycosidic linka e to serine or threonine. The structures

Cited by 13 publications

References 6 publications

Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis

Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis

Identification and structural analysis of the tetrasaccharide NeuAc.alpha.(2.fwdarw.6)Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.fwdarw.3)Fuc.alpha.1.fwdarw.O-linked to serine 61 of human factor IX

Recovery of Intact 2-Aminobenzamide-Labeled O-Glycans Released from Glycoproteins by Hydrazinolysis

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