title: Chemoenzymatic synthesis of multivalent neoglycoconjugates carrying the helminth glycan antigen LDNF.
abstract: Several parasitic helminthes, such as the human parasite Schistosoma mansoni, express glycoconjugates that contain terminal GalNAc beta1-4(Fuc alpha1-3)GlcNAc beta-R (LDNF) moieties. These LDNF glycans are dominant antigens of the parasite and are recognized by human dendritic cells via the C-type lectin DC-SIGN. To study the functional role of the LDNF antigen in interaction with the immune system, we have developed an easy chemoenzymatic method to synthesize multivalent neoglycoconjugates carrying defined amounts of LDNF antigens. An acceptor substrate providing a terminal N-acetylglucosamine was prepared by coupling a fluorescent hydrophobic aglycon, 2,6-diaminopyridine (DAP), to N,N'-diacetylchitobiose. By the subsequent action of recombinant Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase and human alpha1,3-fucosyltransferase VI (FucT-VI), this substrate was converted to the LDNF antigen. We showed that human FucT-VI has a relatively high affinity for the unusual substrate GalNAc beta1-4GlcNAc (LDN), and this enzyme was used to produce micromolar amounts of LDNF-DAP. The synthesized LDNF-DAP was coupled to carrier protein via activation of the DAP moiety by diethyl squarate. By varying the molar glycan:protein ratio, neoglycoconjugates were constructed with defined amounts of LDNF, as was determined by MALDI-TOF analysis and ELISA using an anti-LDNF antibody.
authors.affiliation: Department of Molecular Cell Biology and Immunology, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
authors.forename: Boris
authors.initials: B
authors.lastname: Tefsen
authors.forename: Caroline M W
authors.initials: CM
authors.lastname: van Stijn
authors.forename: Marloes
authors.initials: M
authors.lastname: van den Broek
authors.forename: Hakan
authors.initials: H
authors.lastname: Kalay
authors.forename: Jaco C
authors.initials: JC
authors.lastname: Knol
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jimenez
authors.forename: Irma
authors.initials: I
authors.lastname: van Die
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D006001:Glycoconjugates / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry; D008962:Models, Theoretical; D015394:Molecular Structure; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000947:Antigens, Helminth; D004187:Disaccharides; D006001:Glycoconjugates; C488538:N-acetylgalactosaminyl-1-4-(fucosyl-1,3)-N-acetylglucosamine; D011725:Pyridines; D011994:Recombinant Proteins; C032438:chitobiose; D005647:Fucosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C031692:galactoside 3-fucosyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; D007785:Lactose; C505797:2,6-diaminopyridine