Molecular Immunology 57 (2014) 220–225 Contents lists available at ScienceDirect Molecular Immunology jo u r n al homep age: www.elsevier.com/locate/molimm Short communication Cloning, expression in E. coli and immunological characterization of Par j 3.0201, a Parietaria pollen profilin variant A. Bonura a , A. Trapani a , L. Gulino a , V. Longo a , R. Valenta b , R. Asero c , P. Colombo a,∗ a Istituto di Biomedicina ed Immunologia Molecolare,“Alberto Monroy” del Consiglio Nazionale delle Ricerche, Palermo, Italy b Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, AKH, Vienna, Austria c Allergy Department of the Clinica San Carlo, Paderno Dugnano, Italy a r t i c l e i n f o Article history: Received 4 July 2013 Received in revised form 6 September 2013 Accepted 9 September 2013 Available online 26 October 2013 Keywords: Parietaria pollen Allergen Profilin Recombinant allergen a b s t r a c t Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA con- taining the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of pro- filin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Allergies affect growing numbers of people living in industri- alized countries. The Skin Prick Test (SPT) represents a widely used method to diagnose this kind of pathology although it has the drawback of being performed with natural extracts that are heterogeneous mixtures of several allergenic proteins whose standardization is difficult to achieve. Sensitization to plant pan- allergens (i.e. Profilins, Calcium Binding Proteins, and Lipid Transfer Proteins) poses relevant diagnostic problems as they show highly conserved amino acid sequences that are widely distributed in the plant kingdom making the interpretation of diagnostic test based on natural extracts difficult due to their IgE cross-reactivity. The development of in vitro diagnostic tests based on the use of purified natural and/or recombinant allergens gives a well-defined picture of a patient’s IgE recognition (Hiller et al., 2002), therefore, knowledge of the complete spectrum of allergens contained in ∗ Corresponding author at: Istituto di Biomedicina ed Immunologia Molecolare, “Alberto Monroy” del Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy. Tel.: +39 91 6809535; fax: +39 91 6809548. E-mail address: paolo.colombo@ibim.cnr.it (P. Colombo). an allergenic source and the identification of markers of sensi- tization represents a key issue for a correct diagnosis and for the subsequent prescription of allergen specific immunotherapy vaccines (Mothes et al., 2006; Valenta and Niederberger, 2007). Profilins are small cytosolic proteins found in all eukaryotic cells as part of the cytoskeleton. Plant profilins are usually minor aller- gens (Anto et al., 2012) and cause IgE cross-reactivity not only among botanically unrelated pollens (Valenta et al., 1992) but also between pollens and foods (van Ree et al., 1992) as well as pollen and latex (Ganglberger et al., 2001). Most studies have confirmed the immunological equivalence of profilins from different sources (Ganglberger et al., 2001; van Ree et al., 1992), but some reports found limited cross-reactivity of some specific profilins, such as that from Parietaria, with birch and grass profilins (Asero et al., 2003). Parietaria is one of the most relevant causes of pollen allergy in people living in the Mediterranean basin (D’Amato et al., 2007). About 30% of all allergic subjects living in southern Italy are positive on SPT with Parietaria judaica (Pj) pollen extract (D’Amato, 2000). The composition of allergenic extracts of Pj pollen has been studied by several methods showing the presence of two major allergens belonging to the Lipid Transfer Protein (LTP) family (Par j 1 and Par j 2) (Colombo et al., 2003) as well as the presence of two highly cross-reactive allergens, namely Calcium Binding Protein (Par j 4) 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.09.004