Characterisation of horse dander allergen glycoproteins using amino acid and glycan structure analyses. a mass spectrometric method for glycan chain analysis of glycoproteins separated by two-dimensional electrophoresis.
Separation of horse dander allergens using two-dimensional PAGE resulted in the identification of 16 proteins that react with allergic patient sera. A sensitive method has been developed for analysing the structures of the glycan chains of individual glycoprotein allergens transferred to blots following two-dimensional PAGE, and has allowed the structural identification of the glycan chains of the most abundant isoforms of Equ c 1, a glycosylated horse dander major allergen. The method involves separation of the allergens by two-dimensional PAGE, transfer to polyvinylidene difluoride membranes, release of the glycan chains using peptide N-glycosidase F, permethylation and mass spectrometric analysis of the derivatised glycans. The amino acid compositions of the 16 horse dander allergens separated by two-dimensional PAGE have been determined, allowing the identification of the various isoforms of Equ c 1. These results also confirmed that the two non-glycosylated major allergens, Equ c 2.0101 and Equ c 2.0102, belong to the lipocalin family, and support the idea that these two allergens are most probably isoforms of the same protein. The glycan structures identified using the mass spectrometric method are common biantennary and triantennary glycan chains. These carbohydrate moieties may have a role in the binding of IgE; however, it is more likely that the overall glycoprotein structure involving both the glycan and protein moieties, rather than the structure of the glycan chains alone, is responsible for eliciting allergic responses.
Bulone
V
V
Institute of Pharmacy, University of Oslo, Oslo, Norway. bulone@cermav.cnrs.fr
Rademaker
G J
GJ
Pergantis
S
S
Krogstad-Johnsen
T
T
Smestad-Paulsen
B
B
Thomas-Oates
J
J
D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000581:Amidohydrolases; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000737:chemistry*; D006736:Horses / Q000276:immunology*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D054834:Lipocalins; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000032:analysis*; D020033:Protein Isoforms / Q000737:chemistry
D000485:Allergens; D000596:Amino Acids; C103025:Equ c1 allergen, Equus caballus; D006023:Glycoproteins; D054834:Lipocalins; D011134:Polysaccharides; D020033:Protein Isoforms; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase