Cloning and Expression of the <i>Olea europaea</i> Allergen Ole e 5, the Pollen Cu/Zn Superoxide Dismutase

Abstract

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. Methods: cDNA of Ole e 5 was amplified by nested 3′-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients’ IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). Results: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. Conclusions: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Secretome analysis of novel IgE‐binding proteins from Penicillium citrinum

“…Three different SOD isoforms (Fe SOD, Mn SOD, and Cu/Zn SOD) have been identified depending on the metal cofactor used. Ole e 5 from Olea europaea was the first Cu/Zn SOD identified as a pollen allergen [31]. In fungi, only Asp f 6 (Mn SOD) reacts positively in skin tests, and binds to IgE in allergic bronchopulmonary aspergillosis patients [1].…”

Purification and Characterization of a Cu,Zn-SOD from Garlic (Allium sativum L.). Antioxidant Effect on Tumoral Cell Lines

“…Cu,Zn-SOD, which is the most prevalent SOD in plant tissue, has been purified from a variety of plant sources including spinach [12], cabbage [13], maize [14], Nicotiana plumbaginifolia [15], Radix lethospermi [16], Olea europea [17]. Garlic (Allium sativum L.) is an extensively studied medicinal plant with a wide range of pharmacological effects, such as hypolipidemic, antihypertensive, antibacterial, and antitumoral [18].…”

In silico identification of potential new latex allergens

“…Ole e 5 was discovered in 1998 [24] and is the first Cu/Zn superoxide dismutase identified as an allergen in a pollen source. Ole e 5 has an IgE-binding frequency in O. europaea-allergic subjects of about 35% [24,25], and has been proposed [25] as a possible source of crossreactivity, due to its widespread presence in plants. This suggestion [25] agrees well with our data.…”