Primary structure of 21 novel monoantennary and diantennary N-linked carbohydrate chains from alpha(D)-hemocyanin of Helix pomatia

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein alpha(D)-hemocyanin (alpha(D)-Hc) of Helix pomatia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an alpha(D)-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH2, was characterized using H-1-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of alpha(D)-Hc were released with peptide-N-4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH2/Lichrosphere-NH2, and/or Lichrosphere-C-18. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz H-1-NMR spectroscopy. In total, four novel types of antenna were identified, namely, [GRAPHICS] which are all attached to O-2 of alpha Man residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally beta 1,2-xylosylated and alpha-1,6-fucosylated, Man(alpha 1-6)[Man(alpha 1-3)][+/-Xyl(beta 1-2)]Man(beta 1-4)GlcNAc(beta 1-4)[+/-Fuc(alpha 1-6)]GlcNAc.