Allergenicity of Hev b4 major protein is largely determined by its carbohydrate

Abstract

RATIONALE: A lecithinase homolog is the major protein component of Hev b4, a latex allergen that is reactive in skin prick tests. On SDS-PAGE, the molecular weight of the protein appeared significantly higher than that derived from its cDNA. An investigation was undertaken to verify if the discrepancy was due to carbohydrate, for which seven N-glycosylation sites were predicted on the protein sequence. METHODS: The Hev b4 major protein was deglycosylated by trifluoromethane sulfonic acid and protein migration on SDS-PAGE before and after deglycosylation was compared. The cDNA encoding the protein obtained by reverse transcription-PCR was employed for over-expression as fusion protein in Escherichia coli. Western IgE immunoblots of the native and recombinant proteins were performed using sera from latex allergic patients. RESULTS: Carbohydrate detection of Hev b4 blotted on to nitrocellulose confirmed it to be a glycoprotein. On SDS-PAGE, intact native Hev b4 showed an apparent molecular weight of 52 kDa. On the other hand, its deglycosylated form was 40 kDa, this being comparable to the 38.75 kDa mass predicted by its cDNA sequence. Western immunoblots showed that the deglycosylated Hev b4 had lost its ability to bind human IgE although the protein retained recognition to polyclonal anti-Hev b4 IgG. The unglycosylated E. coli recombinant Hev b4 also failed to recognize IgE from latex-allergic patients. CONCLUSIONS: The IgE epitopes of the Hev b4 major protein reside in its carbohydrate moiety that is responsible for the discrepancy between observed and calculated molecular weights.