Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry.
The lecithinase homolog (Hev b 4) from Hevea brasiliensis (Q6T4P0_HEVBR) is an important natural rubber latex allergen. Hev b 4 is a highly glycosylated protein and its carbohydrate moiety has been implicated in the binding of IgE from natural rubber latex allergic patients. The cDNA for Hev b 4 has recently been cloned and sequenced. Here, we have analyzed the post-translational modifications of natural Hev b 4 by liquid chromatography/electrospray ionization-mass spectrometry of tryptic peptides. Seven of the eight potential glycosylation sites were found to be occupied. One site, however, was only partially glycosylated. Asn224 was substituted by complex type N-glycans with fucose and xylose, whereas all other sites carried either oligomannose glycans or a mixture of oligomannose and complex N-glycans. Glycosylation site Asn308, the most C-terminal one of the eight sites, was only found in the non-glycosylated form. The complex type N-glycans apparently form the molecular basis for the immune reaction with patients' sera. A large fraction of Hev b 4 molecules contains two or more complex N-glycans and thus a physiological reaction against these polyvalent allergens on the basis of the carbohydrate is in theory possible. Aside from allowing glycosylation analysis, the mass spectrometric data defined the N-terminal cleavage site of Hev b 4. This study once more demonstrates the outstanding analytical potential of electrospray ionization-mass spectrometry coupled with liquid chromatographic separation.
Kolarich
Daniel
D
Division of Biochemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Altmann
Friedrich
F
Sunderasan
Elumalai
E
D000485:Allergens / Q000737:chemistry*; D052179:Antigens, Plant / Q000737:chemistry*; D001665:Binding Sites; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D028482:Hevea / Q000276:immunology; D013058:Mass Spectrometry; D010740:Phospholipases / Q000737:chemistry*; D010940:Plant Proteins / Q000276:immunology; D011134:Polysaccharides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D012408:Rubber / Q000737:chemistry*
D000485:Allergens; D052179:Antigens, Plant; D006023:Glycoproteins; D010940:Plant Proteins; D011134:Polysaccharides; D012408:Rubber; C510766:Hev b 4 allergen, Hevea brasiliensis; D010740:Phospholipases