Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4.
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 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa 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 characterize the native protein and its recombinant form in relation to IgE binding.


METHODS
The 5'/3' 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.


CONCLUSIONS
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.
Sunderasan
E
E
Biotechnology and Strategic Research Unit, Malaysian Rubber Board, Kuala Lumpur, Malaysia. sunderasan@lgm.gov.my
Bahari
A
A
Arif
S A M
SA
Zainal
Z
Z
Hamilton
R G
RG
Yeang
H Y
HY
D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004342:Drug Hypersensitivity / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006031:Glycosylation; D028482:Hevea / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007840:Latex / Q000276:immunology; D010740:Phospholipases / Q000276:immunology*; D010940:Plant Proteins / Q000276:immunology; D011134:Polysaccharides / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012408:Rubber
D000485:Allergens; D018076:DNA, Complementary; D007840:Latex; D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D012408:Rubber; D010740:Phospholipases