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-gly cans of the glycoprotein alpha(D)-hemocyanin (alpha(D)-Hc) of Helix po matia 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 fract ionation on Lichrosorb-NH2, was characterized using H-1-NMR spectrosco py, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate ch ains of alpha(D)-Hc were released with peptide-N-4-(N-acetyl-beta-gluc osaminyl)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfr actions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosor b-NH2/Lichrosphere-NH2, and/or Lichrosphere-C-18. Purified oligosaccha rides and oligosaccharide-alditols were analyzed using 500/600-MHz H-1 -NMR spectroscopy. In total, four novel types of antenna were identifi ed, namely, [GRAPHICS] which are all attached to O-2 of alpha Man resi dues 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.