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BEEZHOLD, D.

CARVALHO, L. J. C. B.

SOUZA, C. R. B. de

English

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900 Protein & Peptide Letters, 2008, 15, 900-902   0929-8665/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd. Pt2L4 Protein, a Homologue to Hev b 5 from Rubber Tree, May Not Be Responsible for the Cross-Reactions to Cassava Show by People Allergic to Latex Cláudia Regina Batista de Souza1,2,*,#, Donald Beezhold3,4 and Luiz Joaquim C. Branco Carvalho1 1EMBRAPA Recursos Geneticos e Biotecnologia, Brasília-DF, 70770-900, Brazil. 2Current address: La-boratório de Biologia Molecular, Universidade Federal do Pará, Belém-PA, 660075-110, Brazil. 3Guthrie Research Institute, Sayre, Pa., USA. 4Current address: NIOSH, Morgantown, WV, USA Abstract: Pt2L4 is a protein from cassava homologue to Hevb5, a principal allergen from latex. Here we aimed to elucidate immunological relationships between these proteins. Our results revealed that epitopes found in Hev b 5 are not entirely conserved in Pt2L4 which is not recognized by IgE from patients allergic to Hev b 5. Keywords: Allergenicity, Cassava, Glutamic acid-rich protein, Latex-fruit syndrome, Storage root formation. #Author Profile: Cláudia Regina Batista de Souza is a Research Professor at Universidade Federal do Pará (Belém-PA, Brazil). She is a molecular biologist and has experience in plant molecular biology and gene expression regulation. Cláudia’s current research is about genes related to storage root formation in cassava and plant-pathogen interaction, including mechanism of resistance to Fusarium disease in Piper species from Amazon forest. Her research has been supported by grants from competi-tive public research agencies, especially the Brazilian Research Council (CNPq). INTRODUCTION  Pt2L4 is a glutamic acid-rich protein related to secondary growth and storage root formation in cassava (Manihot escu-lenta Crantz) [1, 2] that is 42% identical to Hev b 5 protein from rubber tree [3], a major allergenic agent with a reported prevalence of specific serum IgE of 52% to 92% among al-lergic patients to latex [4, 5]. Less than 1% of the general population is allergic to latex; however the prevalence of latex allergy in higher in people who wear latex gloves at work as sanitary workers (17%) and in people who have suf-fered many surgical operations, like Spina bifida children (50%) [6]. About 21 and 58% of latex-allergic patients show allergenic responses to banana, papaya, avocado and kiwi fruits, also called latex fruit syndrome [7]. Allergy to cassava attributed to cross-reactivity with latex has been reported [8-10].  The molecular basis of allergenic cross-reactivity is the occurrence of homologous proteins (or protein domains) in both the primary sensitizer and the cross-reactive allergenic sources. Homologous proteins share different degrees of sequence identity, similar 3D-structures and common epi-topes recognized by IgE antibodies [11]. Homologues to Hev b 5 protein contain a high glutamic acid and alanine content, pI value in the range of 3.6-3.8 and molecular mass around 18 kDa and they have been found in kiwi fruit [4, 5], sugar beet [12] and cassava [3].  *Address correspondence to this author at the EMBRAPA Recursos Geneti-cos e Biotecnologia, Brasília-DF, 70770-900, Brazil. 2Current address: Laboratório de Biologia Molecular, Universidade Federal do Pará, Belém-PA, 660075-110, Brazil; E-mail:  bsouza@ufpa.br   Cassava storage roots are a staple food in Africa, South America and Asia. People allergic to cassava have been re-ported [8-10]; therefore, the identification of cassava pro-teins with allergenic properties is highly relevant. In this work we aimed to evaluate immunological relationships be-tween Pt2L4 and Hev b 5 proteins. Blots with Pt2L4 protein were tested for cross-reactivity against polyclonal antibodies raised against latex and Hev b 5 protein and human IgE from hypersensitive allergic patients to Hev b 5. A comparative sequence analysis was conducted in order to search the Pt2L4 protein for IgE epitopes identified in Hev b 5 by Beezhold et al. [13].  MATERIAL AND METHODS Plant Material  Plants of the commercial cultivar IAC 12-829 were propagated by stem cuttings and planted in field plots of EMBRAPA – Genetic Resources and Biotechnology and grown for 10 months. Storage roots were harvested and roots of uniform 5 cm diameter were sampled to dissect tissue layers as previously described [2]. Dissected tissue from layer 4 was used in Pt2L4 extraction as previously described [14]. Antibodies Raised Against Latex and Hev b 5 Proteins  Human sera with anti-Hev b 5 IgE, rabbit anti-latex and anti-Hev b 5 IgG were obtained as previously reported by Beezhold et al. [13, 15]. The Hev b 5 reactive IgE serum was pooled from 6 latex allergic patients with positive skin tests to recombinant Hev b 5 at 1 ng/ml or less.  Pt2L4 Protein, an Homologue to Hev b 5 from Rubber Tree Protein & Peptide Letters, 2008, Vol. 15, No. 9    901 Western Blot Analysis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots were performed as described by Beezhold et al. [13].  Sequence Analysis  Sequences of Pt2L4 from cassava (AAM55492) and Hev b 5 from rubber-tree (Q39967) were imported from Swis-sprotein Bank and aligned using Clustal program. RESULTS AND DISCUSSION  Patients who are allergic to latex may exhibit cross-hypersensitivity with foods, including cassava which belongs to the Euphorbiaceae family and it is often used as a staple food in Africa, Asia and Latin America. Cross-reactivity between different foods occurs due allergenic proteins show-ing common IgE epitopes [11].   In this work we used IgG antibodies raised against latex and Hev b 5 protein and IgE from patients allergic to Hev b 5 in order to identify cross-reactivity between Pt2L4 protein from cassava and a major latex allergen. These proteins have unusually high levels of glutamic acid, alanine and proline residues and migrate at 32-36 kDa in SDS-PAGE although they have a predicted molecular weight of 16-18.0 kDa [2, 4, 5]. Differences between predicted and apparent molecular mass is often seen with acidic proteins and has been ob-served by others [16, 17].   Studies revealed that in Western blot experiments Hev b 5-specific antibodies recognize a broad range of protein in the 36- to 100-kD [14, 18] and a similar pattern was also detected in Pt2L4 sample (Fig. 1). Our results show that Pt2L4 protein is recognized by IgG antibodies raised against latex and Hev b 5 protein, but not by human IgE from hyper-sensitive patients allergic to Hev b 5.   The recognition of Hev b5 by sera pooled from Hev b 5-allergic patients has been attributed to 11 different IgE epi-topes [13] which are mostly located in regions of alignment gaps or non-homology with the Pt2L4 protein (Fig. 2). The results obtained here indicate that despite these two proteins         Figure 1. Western blot analysis of Pt2L4 and Hev b 5 proteins probed with different antibodies: A- Rabbit pre immune serum, B- Rabbit IgG raised against latex, C-Rabbit IgG raised against Hev b 5 and D- Human IgE from hypersensitive patients allergic to Hev b 5. Proteins were separated in SDS- PAGE, blotted on a nitrocellulose membrane and probed with antibodies. MWM: molecular weight maker (kDa), 1: Pt2L4 protein, 2: Bovine Serum Albumin (as a control) and 3: Hev b5 protein.                 Figure 2. Sequence alignment between Pt2L4 from cassava (AAM55492) and Hev b 5 from rubber-tree (Q39967) using Clustal program. Dashes are sequence gaps. IgE epitopes identified in Hev b 5 by Beezhold et al. [13] are underlined.             						            Hev b5   1  MASVEVESAATALPKNETPEVTKAEETKTEE—-PAAPPASEQETADATP--EKEEPTAAP Pt2L4    1  MATAEVVTAQTALPEEKPAEEVKVSEIVTEEAAPAVEPVAE-EPKEAEPVAVSEEPKEAD   Hev b5  57  AEPEAPAPETEKAEEVEKIEK-TEEP---APEADQTTPEEKPAEP---------EPVAE- Pt2L4   60  DAPAEVAVETKEVVEVEEAKTVTEEPTVEKTEEEEETPKEETPEPVVVKETPKEEPAAET   Hev b5  103 ---EEPKHETK-ETETEAPA---APAE-GEKPAEEEKPITEAAETATTE--VPVEKTEE Pt2L4   120 VVVEAPKETTEAATEAEAPAPESAPASASETPAEEEVPKEEEGDEKKSEAEVEAEKTE-  902    Protein & Peptide Letters, 2008, Vol. 15, No. 9 de Souza et al. have antigenic similarities, the Pt2L4 may not be responsible for the allergic cross-reactions to cassava showed by people allergic to latex. Other proteins possibly related to this cross-reactivity can include a 42-44 kDa protein similar to Hev b 7 or patatin-like protein [9] and a homologue to prohevein (Hev b 6) [10]. ACKNOWLEDGMENTS  Thanks go to the Brazilian National Research Council (CNPq) for the fellowship grant # 142760/1996-0 to CRBS, to the Inter-American Institute for Agriculture Cooperation (IICA) for grants # 244/01 and 305/00 (for CRBS) and to EMBRAPA–Genetic Resources and Biotechnology (grant # 03.1998.027 to LJCBC). REFERENCES [1] de Souza, C.R.B., Carvalho, L.J.C.B. and Cascardo J.C.M. (2004) Protein Pept. Lett., 11, 577-582. [2] de Souza, C.R.B., Carvalho. L.J.C.B., de Almeida E.R.P. and Gan-der E.S. (2006) Protein Pept. Lett., 13, 653-657. [3] de Souza, C.R.B., Carvalho, L.J.C.B., de Almeida, E.R.P. and Gander E.S. (2004) In: Proceedings of the Sixth International Sci-entific Meeting of Cassava Biotechnology Network. Cali, Colom-bia. S5-06. [4] Slater, J.E., Vedvick, T., Arthur-Smith, A., Trybul, D.E. and Kek-wick, R.G.O. (1996) J. Biol. Chem., 271, 25394–25399. [5] Akasawa, A., Hsieh, L.S., Martin, B.M., Liu, T. and Lin, Y. (1996) J. Biol. Chem., 271, 25389–25393. [6] Sánchez-Monge, R.;  Salcedo, G. (2006) In: Can cross-reactivity studies enable generic allergy? (Gilissen, L.J.E.J., Wichers, H.J., Savelkoul, H.F.J. and Bogers, R.J. Eds.), Springer Verlag, New York, pp. 45-56. [7] Blanco, C. (2003) Current Allergy and Asthma Reports, 3, 47-53. [8] Galvao, C.E.S., Iwai, L.K., Andrade, M.E.B., Kalil J.and Morato, F.F. (2003) J. Allergy Clin. Immunol., 113:S61. [9] Gaspar, A., Neto-Braga, C., Pires, G., Murta, R., Morais-Almeida, M. and Rosado-Pinto, J. (2003) Allergy, 58, 683-684. [10] Ibero, M., Castillo, M.J. and Pineda, F. (2007) J. Investig. Allergol. Clin. Immunol., 17, 409-412. [11] Aalberse, R.C., Akkerdaas, J. and Van Ree, R. (2001) Allergy, 56, 478-490. [12] Fowler, M.R., Gartland, J., Norton, W., Slater, A., Elliott, M.C. and Scott N.W. (2000) J. Exp. Bot., 51, 2125–2126. [13] Beezhold, D.H., Hickey, V.L., Slater, J.E. and Sussman G.L. (1999) J. Allergy Clin. Immunol., 103, 1166–1172. [14] de Souza, C.R.B., Carvalho, L.J.C.B., de Almeida, E.R.P. and Gander, E.S. (2002) Plant Foods Hum. Nutr., 57, 353-363. [15] Beezhold, D.H., Hickey, V.L. and Sussman, G.L. (2001) J. Allergy Clin. Immunol., 107, 1069–1076. [16] Rasmussen, H.B. (1993) APMIS Suppl., 39, 1-28. [17] Hames, B.D. (1981) In: Gel Electrophoresis of Proteins (Hames B.D. and Rickwood D.) pp. 1-91. Oxford University Press, New York. [18] Sutherland, M.F., Drew, A., Rolland, J.M., Slater, J.E., Supioglu, C. and O’Hehir, R.E. (2002) Clin. Exp. Allergy, 32, 583–589.    Received: January 01, 2008 Revised: May 15, 2008 Accepted: May 22, 2008 

Pt2L4 protein, a homologue to Hev b 5 from rubber tree, may not be responsible for the cross-reactions to cassava show by peope allergic to latex.

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Pt2L4 protein, a homologue to Hev b 5 from rubber tree, may not be responsible for the cross-reactions to cassava show by peope allergic to latex.

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