Abstract
The gut-associated excretory antigen CAA (circulating anodic antigen) from adult Schistosoma mansoni worms was isolated by immunoaffinity chromatography. Amino acid analysis following alkaline borohydride treatment indicated that CAA is a glycoprotein, O-glycosylated at Thr. The primary structure of the released O-glycan moiety was investigated by one- and two-dimensional, homo- and heteronuclear 1H and 13C NMR spectroscopy. It was found that the major carbohydrate chains have a novel polysaccharide structure, consisting of a branched disaccharide repeating unit containing 2-acetamido-2-deoxy-beta-D- galactopyranose (beta-D-Galp-NAc) and beta-D-glucopyranuronic acid (beta-D-GlcpA). [formula: see text] The major antigenic character of CAA arises from this novel polysaccharide, which was shown to be an absolutely specific diagnostic marker in schistosomiasis. The cross-reactivity of CAA with anti-CCA (circulating cathodic antigen) monoclonal antibodies is caused by the presence of a small amount of O-linked CCA-poly-Lewis x carbohydrate chains on the CAA protein chain.
Highlights
From the Wijvoet Center, Department of Bio-organic Chemistry, Utrecht University, I? 0.Box 80.075, NL-3508 T B Utrecht, The Netherlands and the §Departmenot f Parasitology, Faculty of Medicine, University of Leiden, I! 0
The primarsytructureof the released0-glycanmoi- released into the circulation of the host by the schistosome, etywasinvestigated by one-andtwo-dimensional, upon regurgitating the undigested contentosf the gut.For the homo- and heteronuclear‘H and lSCNMR spectroscopy. purpose of immunodiagnosis of active schistosomiasis in sero
The500amamlrkeedaansltciu,unureresvpdihenoaogftspAp4-hnW0ia5tAtarn-sotmerpi-hc.leahTnbloyehrlleoCepadAhcoAestmipcchoAaanctbcieeda1n. s2tFr0aoa-rt1isodBuni1br0eswt-crAtaa.staeAn,rtfeittgaehderencaaodglbeaostiroencrsdbtteiavoanencl,dsoeitpfa--nwda-sAtarnoMdscX6oT0p-0y6,0MU0Htsrpze ce'HhcttrNoUMmnRei vteesrrpssei(ctBytri)ajavtowaeetprrCeoebrneetcetoerr,mdDpeedepornaartBtumrreuenkofte r3oA0f0NMKMX.R-7550-0sMpaeHncdzferent antigen preparations(AWA-trichloroaceticacid, intact and alka- 13CNMR spectra wererecorded on a Bruker AC-300 spectrometer (Delineborohydride-treatedpurified circulating anodic antigen (CAA)) in phosphate-bufferedsaline partment of Organic Chemistry, Utrecht Universitya)t a probe temperwere coated ontothe ELISA plateand detected with the alkalinephos- ature of 300 K
Summary
Bijvoet Center,Dept. coherence; HOHAHA, homonucleaHr artmann-Hahn;mAb, monoclonal of Bio-organic ChemistryU, trecht UniversityP,. 0.Box 80.075, NL-3508 antibody;MS,massspectrometry;NOESY,nuclearOverhauser en-. S2tFr0aoa-rt1isodBuni1br0eswt-crAtaa.staeAn,rtfeittgaehderencaaodglbeaostiroencrsdbtteiavoanencl,dsoeitpfa--nwda-sAtarnoMdscX6oT0p-0y6-,0MU0Htsrpze ce'HhcttrNoUMmnRei vteesrrpssei(ctBytri)ajavtowaeetprrCeoebrneetcetoerr,mdDpeedepornaartBtumrreuenkofte r3oA0f0NMKMX.R-7550-0sMpaeHncdzferent antigen preparations(AWA-trichloroaceticacid, intact and alka- 13CNMR spectra wererecorded on a Bruker AC-300 spectrometer (Delineborohydride-treatedpurified CAA) in phosphate-bufferedsaline partment of Organic Chemistry, Utrecht Universitya)t a probe temperwere coated ontothe ELISA plateand detected with the alkalinephos- ature of 300 K. The HOHAHA spectrum of the Bio-Gel P-2 V, fraction in 'H,O was solution was adjusted to pH 6.0 with formic acid and transferred toa recorded using an MLEV-17 mixing sequence of 120 ms. Chondroitin ABCLyase Digestion-AWA-trichloroacetic acid (2 mg) 'H-'H two-dimensional scalar shift-correlatedspectroscopy (COSY)was was dissolved in 0.1 ml of 40 mM TridHCl, pH 8.0, containing 50 mM carried out usinga spectral width of 4032 Hz in each dimension. 0-linked immunogenic carbohydrates,which in a released form fail to bind to the microtitration plate
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