Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.).
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).
Department of Pharmacy, University of Oslo, Norway.
B M
BM
Nilsen
K
K
Sletten
B S
BS
Paulsen
M
M
O'Neill
H
H
van Halbeek
D000485:Allergens; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D001216:Asparagine / Q000378:metabolism; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D006026:Glycoside Hydrolases / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010940:Plant Proteins; D010944:Plants; D011058:Pollen / Q000276:immunology; D011487:Protein Conformation; D011817:Rabbits
D000485:Allergens; D052179:Antigens, Plant; D000939:Epitopes; D006023:Glycoproteins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D010940:Plant Proteins; C067027:allergen Art v II protein, Artemisia vulgaris L.; D001216:Asparagine; D006026:Glycoside Hydrolases; D003488:Cyanogen Bromide