Two Novel Types of O-Glycans on the Mugwort Pollen Allergen Art v 1 and Their Role in Antibody Binding

Abstract

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz 1 H and 13 C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short ␤1,6-galactan core, which is substituted by a variable number (5-28) of ␣-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) ␤-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp 4 motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with ␣-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked ␤-arabinose residues appeared to react with the antibodies. Mono-␤-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins.

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

“…For characterization of the polysaccharide moiety, the protein backbone was hydrolyzed with 0.22 M BaOH 2 at 100°C for 6 h (11,17). The alkaline hydrolyzate was neutralized with acetic acid and fractionated over Sephadex G15 using 1% acetic acid as the eluent.…”

“…Short arabinan chains linked to Hyp were detected by liquidchromatography with mass spectrometric detection after alkaline hydrolysis of the protein (11,17) and dansylation (18). In this case, alkaline hydrolysis was followed by neutralization with sulfuric acid in the presence of sodium acetate buffer.…”

“…A rabbit antiserum recognizing patches of ␤-arabinosyl residues was available from a previous Art v 1 study and used as described previously (11). Aspergillus niger ␣-arabinofuranosidase was purchased from Megazyme (Wicklow, Ireland).…”

“…Although the molecular weight of Art v 1 is 13-16 kDa, it is migrating between 24 -28 kDa in SDS-PAGE, which results from this rigid and glycosylated C-terminal domain (11). SDS-PAGE and IgE immunoblot experiments using ragweed pollen extract revealed the presence of a glycoprotein similar in size and migrating with the same diffuse double band pattern as Art v 1 (1,2).…”

“…In mugwort pollen, the major allergen is Art v 1, a protein with a globular domain homologous to thionins (or defensins) and a hydroxyproline-rich extensively glycosylated moiety (10,11). Although the molecular weight of Art v 1 is 13-16 kDa, it is migrating between 24 -28 kDa in SDS-PAGE, which results from this rigid and glycosylated C-terminal domain (11).…”