Primary Structure of 21 Novel Monoantennary and Diantennary N‐Linked Carbohydrate Chains from α_D‐Hemocyanin of Helix Pomatia

Abstract

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein a,-hemocyanin (a,-Hc) of Helix pnmutia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an a,-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH,, was characterized using 'H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of a,,-Hc were released with peptide-N4-(Nacetyl-P-glucosaminy1)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-NH,/ Lichrosphere-NH2, and/or Lichrosphere-C,,. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 'H-NMR spectroscopy. In total, four novel types of antenna were identified, namely, 3MeGalwl-6)\ \ \ 3MeGal(~1-3)GalNAc(~1-4)GlcNAc(~1-4MeGal(~I-3)GalNAc(/j'1-4)GlcNAc(~l-which are all attached to 0 -2 of aMan residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally P-1,2-xylosylated and a-I ,6-fucosylated, ][? Xyl(p1-2)]Man(jl1-4)GlcNAc(~1-4) [ 2 Fuc( a 1 -6)] GlcNAc.

Identification and Characterization of Keyhole Limpet Hemocyanin N-Glycans Mediating Cross-reactivity with Schistosoma mansoni

“…This monosaccharide constituent has already been detected in our previous study (21). N-Glycans comprising (␤1-2)-linked xylose are also present in hemocyanins of Helix pomatia (49,50) and Lymnea stagnalis (51). In contrast to KLH, however, xylosylated glycans have been reported to represent major carbohydrate constituents of these molecules.…”

Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non‐specific immunostimulatory effects

“…Gastropod hemocyanins have a carbohydrate content of 2-9% w/w, with mannose being the major monosaccharide found in these structures [24][25][26][27]. To stabilize CCH structure, we oxidize its sugar moieties with sodium periodate in order to induce internal cross-linking within the protein.…”

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds

“…The (α1-3)-fucosylated and (β1-2)-xylosylated N-glycans have been repeatedly reported to be immunogenic in mammals [12][13][14][15][16][17] and in fact (β1-2)-linked Xyl and (α1-3)-linked Fuc residues have been identified as the epitope structures for IgE binding [18,19] in patients allergic to tree and grass pollens. The vast potential for cross-reaction of antibodies with one or both of these carbohydrate determinants is further extended by the Table 5 Survey of N-glycans obtained from gpMuc, together with their relative amounts occurrence of the same epitope structures on glycoproteins of insects, molluscs and parasitic worms [20][21][22]. The suggestion of a contribution of (β1-2)-Xyl and (α1-3)-Fuc residues to cross-reaction mechanisms is further supported by the fact that anti-horseradish peroxidase and the anti-core (α1-3)-Fuc monoclonal YZ1/2.23 antibody bind to pollen [16].…”