Occurrence of Lewis a Epitope inN-Glycans of a Glycoallergen, Jun a 1, from Mountain Cedar (Juniperus ashei) Pollen

Abstract

We have determined the structures of N-glycans linked to major allergens in the mountain cedar (Juniperus ashei) pollen, Jun a 1. First, two kinds of the pollen glycoallergen (Jun a 1-A and Jun a 1-B) were purified from partially purified Jun a 1 by cation exchange chromatography. The N-glycans were liberated by hydrazinolysis from the two glycoallergens and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC from Jun a 1-A and Jun a 1-B respectively. The structures were determined by a combination of exo-and endo-glycosidase digestions, two dimensional sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS) analysis. Structural analysis indicated that Lewis a epitope (Gal1-3(Fuc1-4)GlcNAc1-) occurs in the N-glycans of the pollen allergens.

A Unique β1,3-Galactosyltransferase Is Indispensable for the Biosynthesis of N-Glycans Containing Lewis a Structures in Arabidopsis thaliana

“…Given the complex architecture of the GALT1 protein, it is also possible that structural constraints permit the enzyme to act only on a small subset of endogenous glycoproteins. This is substantiated by the fact that until now only few purified plant glycoproteins have been found to carry Le a epitopes (Fitchette-Lainé et al, 1997;Melo et al, 1997;Kimura et al, 2005).…”

The Role of Protein Glycosylation in Allergy

“…The very large Lewis a type structures are rarely found on soluble allergens and are currently thought to play only a minor, if any, role in IgE binding even though many plant foods (apple, banana, celery, onion, orange, pear, and strawberry) contain large amounts of these near-mammalian N-glycans [20]. The cedar pollen allergens Cry j 1 and Jun a 1 are the only allergens known to contain Lewis a antennae [31, 32]. Complex-type glycans without terminal galactose/fucose comprise the most abundant species in most (food) plants.…”

Glycoform Analysis ofN-Glycans Linked to Glycoproteins Expressed in Rice Culture Cells: Predominant Occurrence of Complex TypeN-Glycans

“…On the other hand, peak e was not digested by this exoglycosidase, suggesting that GlcNAc residues might be replaced by other sugar molecules. Since 2D-sugar chain mapping analysis 17) and ESI-MS suggested that peaks d and peak e might harbor Lewis a epitopes like N-glycans linked to Jun a1 and Cry j1, two major cedar pollen allergens, 15,20) these sugar chains were treated with a mixture of 1-3/4 fucosidase and lacto Nbiosidase to confirm the occurrence of the Lewis a epitope unit (Gal1-3(Fuc1-4)GlcNAc1-). As shown in Fig.…”

“…1, N-glycan-containing fractions, indicated by a horizontal bar (elution time: 35-60 min), were pooled and evaporated to dryness. Since it has been found that almost all N-glycans linked to plant glycoproteins are eluted after M3FX on RP-HPLC, [13][14][15][18][19][20] the pooled fractions indicated by the bars were used for further structural analysis of Nglycans. After the pooled fractions were concentrated to dryness in vacuo, the resulting residue was dissolved in 20 mM Tris-HCl buffer, pH 7.8, containing 0.1 M NaCl, 5 mM CaCl 2 , and 5 mM MgCl 2 (5 ml), and put on a Con A-Sepharose 4B column (2:0 Â 25 cm) equilibrated with the same buffer.…”

“…Digestion of PA-sugar chains with jack bean -mannosidase, Aspergillus -1,2-mannosidase, diplococcal -N-acetylglucosaminidase, -1,3/4-fucosidase, and Lacto-N-biosidase were done using about 200 pmol of PA-sugar chains under the conditions described in previous reports. 14,15) The reactions were stopped by boiling the mixtures for 3 min. After centrifugation at 15,000 rpm for 10 min, the resulting glycosidase-digests were analyzed by SF-HPLC using an Asahipak NH2P-50 column.…”