Abstract
The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).
Highlights
The glycoprotein allergen Art u 11, from the pollen of mugwort (Artemisia vulgarisL.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides
The glycosyl composition andthe high affinity for concanavalin A suggest that at least part of the carbohydrate structure of Art u I1 consists of Asn-linked oligosaccharides of the high-mannose type
1.0 :: am all classes of Asn-linked oligosaccharides (20)T. he extent of enzymatic deglycosylatiownas monitoredby SDS-PAGE(Fig. l ), and it was found that Art u I1 in its native conformation could not be deglycosylatedwith Endo H or PNGase F
Summary
Deglycosylation of Art u 11 with PNGase F and Endo H Glycosyl composition analysis of Art u I1 establishedthe presence of mannose, N-acetylglucosamine, glucose, and galactose intheratios 13.4:3.2:1.3:1.0 (6). Isolation of Asn-linked Oligosaccharides-The products obtained by PNGase F treatment of Art u I1 were fractionated on a column of Bio-Gel P-4.Material containingcarbohydrate eluted from the column in two peaks (Fig. 2) and was well separated from glycerol.Glycerol is present in commercial preparations of PNGase F. Characterizationof Carbohydrate in Pool A-Glycosyl com- 3) These H-1 signals havebeenassigned in the ‘HNMR position analysis of Pool A showed the presence of mannose, spectra of complex Asn-linked oligosaccharides(23),but not galactose, arabinose, and glucose in the ratios 6.41.6:l.Ol.O. in the ‘H NMR spectra of the high-mannosetype.High-. The H-l signals of the GlcNAc-1residue are not present in the ‘H NMR spectra of high-mannose oligosaccharides releasebdy Endo H.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have