title: Targeted identification of a unique glycan epitope of Schistosoma mansoni egg antigens using a diagnostic antibody.
abstract: The eggs of Schistosoma mansoni express a plethora of glycoconjugate antigens. A specific subset of these antigens can be detected in the serum or urine of infected individuals by a diagnostic sandwich ELISA using the anti-carbohydrate monoclonal antibody (mAb) 114-4D12-A [Nourel Din MS, Nibbeling R, Rotmans JP, Polderman AM, Krijger FW, Deelder AM. Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1994;50:585-94]. We used affinity chromatography to isolate the 114-4D12-binding glycoprotein subset from soluble egg antigens (SEA) of S. mansoni. SEA and the isolated SEA-subset (SEA-4D12) were subjected to reductive beta-elimination and hydrazinolysis to release intact glycans and glycan fragments, respectively, from the protein backbones. The released glycans were characterised by matrix-assisted laser-desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS), liquid-chromatography (LC)-MS and gas chromatography (GC)-MS linkage analysis. Glycans released by reductive beta-elimination from SEA-4D12 were larger and more heavily fucosylated than glycans released from SEA. Most SEA-4D12 glycans contained a branched O-glycan core structure carrying up to 4 N-acetylhexosamines per chain which were substituted with maximum 12 fucose residues. Hydrazinolysis of SEA-4D12 resulted in the release of fucosylated antenna fragments. After 2-aminobenzamide (2AB)-labelling these fragments were subjected to 114-4D12-affinity purification. Normal phase (NP)-LC analysis of the flow-through and retained fractions indicated that the Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1- element forms the epitope of mAb 114-4D12. Most O-glycans from SEA-4D12 contain this structural element. Epitope-bearing N-glycans have not been found. In terms of abundance in total SEA, only a minority of all glycans possesses the epitope. This multifucosylated motif has so far only been found in schistosomes, providing a structural basis for the high specificity of the diagnostic antibody.
authors.affiliation: Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Marjolein L M
authors.initials: ML
authors.lastname: Robijn
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: Louise
authors.initials: L
authors.lastname: Royle
authors.forename: Rudolf
authors.initials: R
authors.lastname: Geyer
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Hokke
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000947:Antigens, Helminth / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D006834:Hydrazines / Q000378:metabolism; D008969:Molecular Sequence Data; D010063:Ovum / Q000276:immunology*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology*; D012550:Schistosoma mansoni / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000909:Antibodies, Helminth; D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D000939:Epitopes; D006023:Glycoproteins; D006834:Hydrazines; D011134:Polysaccharides; C029424:hydrazine; D005643:Fucose