Major mountain cedar allergen, Jun a 1, contains conformational as well as linear IgE epitopes.
We have previously identified four linear IgE epitopes on Jun a 1, the dominant allergen in mountain cedar pollen, and mapped these to the surfaces of a molecular model and to the crystal structure of this glycoprotein. The aim of the present study was to determine if Jun a 1 also displays conformational IgE epitopes. Jun a 1 was denatured by heating at 75 degrees C for 1h, exposure to 6M guanidine or by reductive alkylation in the presence and absence of guanidine. The effects of these manipulations on the binding to IgE from patients with mountain cedar hypersensitivity was evaluated by an ImmunoCAP inhibition assay, using Jun a 1-specific caps. Treatment-associated changes in the 3D-structure were assessed by dynamic light scattering and CD spectroscopy. IgE binding to native Jun a 1 was inhibited 92+/-9% by soluble native protein and 92+/-9% by reduced and alkylated Jun a 1. However, the capacity of Jun a 1 to inhibit the binding of IgE antibodies was significantly diminished upon denaturation by heat, guanidine alone, or reduction and alkylation in guanidine, compared to native Jun a 1. Reductive alkylation treatment under denaturing conditions also increased the Stoke's radius, suggesting that the protein was partially unfolded. Analysis of the circular dichroism (CD) spectra suggested that heating and treatment with guanidine caused a loss of alpha-helical structure. Guanidine also caused an increase in random coil structure. Thus, at least a portion of the anti-Jun a 1 IgE antibodies produced by allergic humans recognize conformational epitopes and it is likely that some of these epitopes reside in alpha-helical structures of Jun a 1.
Varshney
Shikha
S
Department of Pediatrics, Child Health Research Center, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0366, USA.
Goldblum
Randall M
RM
Kearney
Christopher
C
Watanabe
Masanao
M
Midoro-Horiuti
Terumi
T
D000485:Allergens / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D052179:Antigens, Plant; D002942:Circular Dichroism; D018604:Epitope Mapping; D019791:Guanidine / Q000494:pharmacology; D006361:Heating; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D012542:Scattering, Radiation
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D016056:Immunodominant Epitopes; D010940:Plant Proteins; C121951:jun a 1 protein, Juniperus ashei; D007073:Immunoglobulin E; D019791:Guanidine