A novel Gal(beta1-4)Gal(beta1-4)Fuc(alpha1-6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet haemocyanin (KLH) N-glycans.
KLH (keyhole limpet haemocyanin), the oxygen-carrying molecule of the marine snail Megathura crenulata, is often used as an adjuvant or as a hapten carrier for immunizations with peptides, oligosaccharides or other low-molecular-mass organic compounds. KLH exhibits several carbohydrate determinants, at least some of which are immunogenic: it shares an antigenic Fuc(alpha1-3)GalNAc-determinant with schistosomes and contains unique Gal-(beta1-6)Man-structural motifs on its N-glycans. This study reveals the presence of N-glycans with unusual +/-Gal(beta1-4)Gal(beta1-4)Fuc- units (alpha1-6)-linked to the reducing end N -acetylglucosamine residue. The following novel structures of KLH N-glycans were deduced by linkage analysis, exoglycosidase digestion, matrix-assisted laser-desorption ionization-tandem MS and nano-LC-ESI-IT-MS (where LC stands for liquid chromatography, ESI for electrospray ionization and IT for ion trap): Man(alpha1-6)[+/-Man(alpha1-3)]Man(beta1-4)GlcNAc(beta1-4)[Gal(beta1-4)Fuc(alpha1-6)]GlcNAc and Man(alpha1-6)Man(beta1-4)GlcNAc(beta1-4)[Gal(beta1-4)Gal(beta1-4)Fuc(alpha1-6)]GlcNAc. The Gal(beta1-4)Fuc- and Gal(beta1-4)Gal(beta1-4)Fuc- core modifications are expected to be immunogenic, similar to other non-mammalian-type core modifications, and to contribute to the immunostimulatory properties of KLH.
Wuhrer
Manfred
M
Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Robijn
Marjolein L M
ML
Koeleman
Carolien A M
CA
Balog
Crina I A
CI
Geyer
Rudolf
R
Deelder
André M
AM
Hokke
Cornelis H
CH
D000117:Acetylglucosamine / Q000032:analysis*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D005643:Fucose / Q000032:analysis*; D005690:Galactose / Q000032:analysis*; D006433:Hemocyanins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D011134:Polysaccharides; D005643:Fucose; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D000117:Acetylglucosamine; D005690:Galactose