Abstract - Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4


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	Publications of MRB Latest Publications MRB Digest Journal of Rubber Research 3^rd Qtr 2008 Vol 11(3) 2^nd Qtr 2008 Vol 11(2) 1^st Qtr 2008 Vol 11(1) 4^th Qtr 2007 Vol 10(4) 3^rd Qtr 2007 Vol 10(3) 2^nd Qtr 2007 Vol 10(2) 1^st Qtr 2007 Vol 10(1) 1^st Qtr 2006 Vol 9(1) 2^nd Qtr 2006 Vol 9(2) 3^rd Qtr 2006 Vol 9(3) 4^th Qtr 2006 Vol 9(4) Submit paper/article Order Form	Abstract Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4 E. Sunderasan, A. Bahari, S. A. M. Arif, Z. Zainal, R. G. Hamilton and H. Y. Yeang Clinical and Experimental Allergy 2005;35:1490-1495 Background Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognised by the International Union of Immunological Societies, the 53-55kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. Objective We sought to clone the transcript encoding the Hev b 4 major protein, and characterise the native protein and its recombinant form in relation to IgE binding. Methods The 50/30 rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. Results The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. Conclusion The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA. back
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