Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a1

Background The family Cupressaceae is a relevant source of allergens that c auses winter respiratory allergies. Cloning and sequencing the major antige n of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients. Objective To obtain a full-length complementary DNA for Cup a 1, the major allergen of Cupressus arizonica pollen. It was cloned and sequenced and the recombinant protein was expressed. Methods Messenger RNA from Cupressus arizonica pollen was obtained and the Cup a 1 sequence was established using a 3'-RACE system and primers based o n the N-terminal amino acid sequence. Recombinant Cup a 1 was cloned in pBl uescript and expressed in a glycosylated form in rabbit reticulocytes. The cDNA was subcloned in pGEX-5X-1 and expressed in Escherichia coli as a fusi on protein with GST. Results Recombinant Cup a 1 is highly homologous with the major allergens o f mountain cedar (Jun a I), Japanese cypress (Cha o 1) and Japanese cedar ( Cry j 1). Cup a 1 contains three potential N-glycosylation sites that are d ifferent from those found in Jun a 1 and Cry j 1. The cloned protein contai ns a pectate lyase active site identical to these of Cry j 1 and Jun a 1. T he IgE from patients' sera recognizes recombinant Cup a 1, and this reactiv ity is higher with the glycosylated protein. Conclusion Cup a 1 has been cloned and sequenced. As expected, the high deg ree of homology with Cha o 1, Jun a 1 and Cry j 1 explains the cross-reacti vity of conifer pollens. Different IgE reactivity with the glycosylated and non-glycosylated protein suggests the importance of carbohydrate moieties in the IgE binding site.