Studies on Pf332, a major Plasmodium falciparum blood-stage antigen, have largely been hampered by the cross-reactive nature of antibodies generated against the molecule due to its high content of repeats, which are present in other malaria antigens. We previously reported the identification of a conserved domain in Pf332 with a high degree of similarity to the Duffy-binding-like (DBL) domains of the erythrocyte-binding-like (EBL) family. We here describe that antibodies towards Pf332-DBL are induced after repeated exposure to P. falciparum and that they are acquired early in life in areas of intense malaria transmission. Furthermore, a homology model of Pf332-DBL was found to be similar to the structure of the EBL-DBLs. Despite their similarities, antibodies towards Pf332-DBL did not display any cross-reactivity with EBL-proteins as demonstrated by immunofluorescence microscopy, Western blotting, and peptide microarray. Thus the DBL domain is an attractive region to use in further studies on the giant Pf332 molecule. 1. Introduction Plasmodium falciparum malaria is a major human disease that accounts for 1–2 million deaths annually [1]. The disease affects predominantly children under the age of five, as older children and adults living in malaria endemic areas become immune against severe forms of the disease after being repeatedly exposed to the parasite. During blood-stage development, P. falciparum parasites invade red blood cells (RBC) and cause them to sequester from the blood circulation by adhering to host endothelial cells. Both invasion and sequestration are central to the pathogenesis of malaria, and they require an adhesive cysteine-rich domain referred to as the Duffy-binding-like (DBL) domain. The DBL domains are present in two different protein families; the erythrocyte-binding-like (EBL) family of invasion proteins and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence proteins [2, 3]. We recently reported the identification of a previously unknown domain in the P. falciparum antigen 332 with a high degree of similarity to the DBL domains of the EBL family [4]. Most EBL members are transmembrane proteins, which are expressed in schizonts and localize to merozoite micronemes from where they are released prior to or during host cell invasion [5]. In contrast, Pf332 is expressed in trophozoites and is later on cotransported together with the surface-destined PfEMP1 in parasite-induced membrane structures, referred to as Maurer’s clefts [6, 7]. In schizonts, Pf332 can be found in association
References
[1]
WHO, World Malaria Report 2008. Geneva, Switzerland.
[2]
J. H. Adams, P. L. Blair, O. Kaneko, and D. S. Peterson, “An expanding ebl family of Plasmodium falciparum,” Trends in Parasitology, vol. 17, no. 6, pp. 297–299, 2001.
[3]
J. H. Adams, B. K. L. Sim, S. A. Dolan, X. Fang, D. C. Kaslow, and L. H. Miller, “A family of erythrocyte binding-proteins of malaria parasites,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 15, pp. 7085–7089, 1992.
[4]
K. Moll, A. Chêne, U. Ribacke et al., “A novel DBL-domain of the P. falciparum 332 molecule possibly involved in erythrocyte adhesion,” PLoS ONE, vol. 2, article e477, no. 5, 2007.
[5]
B. K. L. Sim, T. Toyoshima, J. D. Haynes, and M. Aikawa, “Localization of the 175-kilodalton erythrocyte binding antigen in micronemes of Plasmodium falciparum merozoites,” Molecular and Biochemical Parasitology, vol. 51, no. 1, pp. 157–159, 1992.
[6]
M. Haeggstr？m, F. Kironde, K. Berzins, Q. S. Chen, M. Wahlgren, and V. Fernandez, “Common trafficking pathway for variant antigens destined for the surface of the Plasmodium falciparum-infected erythrocyte,” Molecular and Biochemical Parasitology, vol. 133, no. 1, pp. 1–14, 2004.
[7]
K. Hinterberg, A. Scherf, J. Gysin et al., “Plasmodium falciparum: the Pf332 antigen is secreted from the parasite by a brefeldin a-dependent pathway and is translocated to the erythrocyte membrane via the maurer's clefts,” Experimental Parasitology, vol. 79, no. 3, pp. 279–291, 1994.
[8]
D. Mattei, K. Berzins, M. Wahlgren et al., “Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens,” Parasite Immunology, vol. 11, no. 1, pp. 15–29, 1989.
[9]
D. Mattei and A. Scherf, “The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages,” Memorias do Instituto Oswaldo Cruz, vol. 87, pp. 163–168, 1992.
[10]
D. Mattei and A. Scherf, “The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes,” Gene, vol. 110, no. 1, pp. 71–79, 1992.
[11]
G. Winter, S. Kawai, M. Haeggstr？m et al., “SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes,” Journal of Experimental Medicine, vol. 201, no. 11, pp. 1853–1863, 2005.
[12]
J. Iqbal, P. Perlmann, B. M. Greenwood, and K. Berzins, “Seroreactivity with the Plasmodium falciparum blood stage antigen Pf332 in adults and children from malaria-endemic regions,” Clinical and Experimental Immunology, vol. 94, no. 1, pp. 68–74, 1993.
[13]
A. Kulane, A. B. Siddique, J. L. Sarthou et al., “Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332,” American Journal of Tropical Medicine and Hygiene, vol. 61, no. 1, pp. 141–148, 1999.
[14]
N. Ahlborg, D. Haddad, A. B. Siddique et al., “Antibody responses to the repetitive Plasmodium falciparum antigen Pf332 in humans naturally primed to the parasite,” Clinical and Experimental Immunology, vol. 129, no. 2, pp. 318–325, 2002.
[15]
Q. J. Chen, A. Heddini, A. Barragan, V. Fernandez, S. F. A. Pearce, and M. Wahlgren, “The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors,” Journal of Experimental Medicine, vol. 192, no. 1, pp. 1–9, 2000.
[16]
J. S？ding, A. Biegert, and A. N. Lupas, “The HHpred interactive server for protein homology detection and structure prediction,” Nucleic Acids Research, vol. 33, no. 2, pp. W244–W248, 2005.
[17]
PDB (2010) PDB: Protein Data Bank.
[18]
N. H. Tolia, E. J. Enemark, B. K. L. Sim, and L. Joshua-Tor, “Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum,” Cell, vol. 122, no. 2, pp. 183–193, 2005.
[19]
A. Sali, L. Potterton, F. Yuan, H. van Vlijmen, and M. Karplus, “Evaluation of comparative protein modeling by Modeller,” Proteins: Structure, Function and Genetics, vol. 23, no. 3, pp. 318–326, 1995.
[20]
C. Gille and C. Fr？mmel, “STRAP: editor for STRuctural alignments of proteins,” Bioinformatics, vol. 17, no. 4, pp. 377–378, 2001.
[21]
W. DeLano, The PyMOL Molecular Graphics System PyMol 1.3 ed, DeLano Scientific, San Carlos, Calif, USA, 2002.
[22]
R. A. Laskowski, M. W. Macarthur, D. S. Moss, and J. M. Thornton, “PROCHECK: a program to check the stereochemical quality of protein structures,” Journal of Applied Crystallography, vol. 26, pp. 283–291, 1993.
[23]
K. Arnold, L. Bordoli, J. Kopp, and T. Schwede, “The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling,” Bioinformatics, vol. 22, no. 2, pp. 195–201, 2006.
[24]
N. Rasti, F. Namusoke, A. Chêne et al., “Nonimmune immunoglobulin binding and multiple adhesion characterize Plasmodium falciparum-infected erythrocytes of placental origin,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 37, pp. 13795–13800, 2006.
[25]
A. Bolad, S. E. Farouk, E. Israelsson et al., “Distinct interethnic differences in immunoglobulin G class/subclass and immunoglobulin M antibody responses to malaria antigens but not in immunoglobulin G responses to nonmalarial antigens in sympatric tribes living in West Africa,” Scandinavian Journal of Immunology, vol. 61, no. 4, pp. 380–386, 2005.
[26]
A. Dolo, D. Modiano, B. Maiga et al., “Difference in susceptibility to malaria between two sympatric ethnic groups in Mali,” American Journal of Tropical Medicine and Hygiene, vol. 72, no. 3, pp. 243–248, 2005.
[27]
D. Modiano, A. Chiucchiuini, V. Petrarca et al., “Humoral response to Plasmodium falciparum Pf155/ring-infected erythrocyte surface antigen and Pf332 in three sympatric ethnic groups of Burkina Faso,” American Journal of Tropical Medicine and Hygiene, vol. 58, no. 2, pp. 220–224, 1998.
[28]
D. Modiano, V. Petrarca, B. S. Sirima et al., “Different response to Plasmodium falciparum malaria in West African sympatric ethnic groups,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 23, pp. 13206–13211, 1996.
[29]
P. E. Okello, W. van Bortel, A. M. Byaruhanga et al., “Variation in malaria transmission intensity in seven sites throughout Uganda,” American Journal of Tropical Medicine and Hygiene, vol. 75, no. 2, pp. 219–225, 2006.
[30]
J. Normark, D. Nilsson, U. Ribacke et al., “PfEMP1-DBL1α amino acid motifs in severe disease states of Plasmodium falciparum malaria,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 40, pp. 15835–15840, 2007.
[31]
P. F. Beales, B. Brabin, E. Dorman et al., “Severe falciparum malaria,” Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 94, pp. S1–S90, 2000.
[32]
M. E. Molyneux, T. E. Taylor, J. J. Wirima, and A. Borgstein, “Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children,” Quarterly Journal of Medicine, vol. 71, no. 265, pp. 441–459, 1989.
[33]
K. Moll, I. Ljungstr？m, H. Perlmann, A. Scherf, and M. Wahlgren, Eds., Methods in Malaria Research, ATCC, Manassas, Va, USA, 5th edition, 2008.
[34]
N. J. Greenfield, “Using circular dichroism spectra to estimate protein secondary structure,” Nature Protocols, vol. 1, no. 6, pp. 2876–2890, 2006.
[35]
S. K. Singh, R. Hora, H. Belrhali, C. E. Chitnis, and A. Sharma, “Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain,” Nature, vol. 439, no. 7077, pp. 741–744, 2006.
[36]
D. Mattei, K. Berzins, M. Wahlgren et al., “Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens,” Parasite Immunology, vol. 11, no. 1, pp. 15–29, 1989.
[37]
C. Spycher, N. Klonis, T. Spielmann et al., “MAHRP-1, a novel Plasmodium falciparum histidine-rich protein, binds ferriprotoporphyrin IX and localizes to the Maurer's clefts,” Journal of Biological Chemistry, vol. 278, no. 37, pp. 35373–35383, 2003.
[38]
F. K. Glenister, K. M. Fernandez, L. M. Kats et al., “Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum,” Blood, vol. 113, no. 4, pp. 919–928, 2009.
[39]
A. N. Hodder, A. G. Maier, M. Rug et al., “Analysis of structure and function of the giant protein Pf332 in Plasmodium falciparum,” Molecular Microbiology, vol. 71, no. 1, pp. 48–65, 2009.
[40]
N. Ahlborg, B. W. Flyg, J. Iqbal, P. Perlmann, and K. Berzins, “Epitope specificity and capacity to inhibit parasite growth in vitro of human antibodies to repeat sequences of the Plasmodium falciparum antigen Ag332,” Parasite Immunology, vol. 15, no. 7, pp. 391–400, 1993.
[41]
N. Ahlborg, J. Iqbal, L. Bj？rk, S. St？hl, P. Perlmann, and K. Berzins, “Plasmodium falciparum: differential parasite growth inhibition mediated by antibodies to the antigens pf332 and Pf155/RESA,” Experimental Parasitology, vol. 82, no. 2, pp. 155–163, 1996.
[42]
N. Ahlborg, J. Iqbal, M. Hansson et al., “Immunogens containing sequences from antigen Pf332 induce Plasmodium falciparum-reactive antibodies which inhibit parasite growth but not cytoadherence,” Parasite Immunology, vol. 17, no. 7, pp. 341–352, 1995.
[43]
R. Udomsangpetch, J. Carlsson, B. Wahlin et al., “Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens,” Journal of Immunology, vol. 142, no. 10, pp. 3620–3626, 1989.
[44]
K. L. Waller, L. M. Stubberfield, V. Dubljevic et al., “Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton,” Biochimica et Biophysica Acta, vol. 1798, no. 5, pp. 861–871, 2010.
[45]
P. Michon, J. R. Stevens, O. Kaneko, and J. H. Adams, “Evolutionary relationships of conserved cysteine-rich motifs in adhesive molecules of malaria parasites,” Molecular Biology and Evolution, vol. 19, no. 7, pp. 1128–1142, 2002.
[46]
C. Aurrecoechea, J. Brestelli, B. P. Brunk et al., “PlasmoDB:a functional genomic database for malaria parasites,” Nucleic Acids Research, vol. 37, no. 1, pp. D539–D543, 2009.
[47]
H. A. Balogun, N. M. Vasconcelos, R. Lindberg et al., “Immunogenicity and antigenic properties of Pf332-C231, a fragment of a non-repeat region of the Plasmodium falciparum antigen Pf332,” Vaccine, vol. 28, no. 1, pp. 90–97, 2009.
[48]
E. Israelsson, H. Balogun, N. M. Vasconcelos et al., “Antibody responses to a C-terminal fragment of the Plasmodium falciparum blood-stage antigen Pf332 in Senegalese individuals naturally primed to the parasite,” Clinical and Experimental Immunology, vol. 152, no. 1, pp. 64–71, 2008.
[49]
M. J. Gardner, N. Hall, E. Fung et al., “Genome sequence of the human malaria parasite Plasmodium falciparum,” Nature, vol. 419, no. 6906, pp. 498–511, 2002.
[50]
X. Z. Su, V. M. Heatwole, S. P. Wertheimer et al., “The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes,” Cell, vol. 82, no. 1, pp. 89–100, 1995.
