Molecular & Biochemical Parasitology 115 (2001) 41 – 53 Plasmodium iax merozoite surface proteins-3 and-3 share structural similarities with P. iax merozoite surface protein-3 and define a new gene family  Mary R. Galinski a, *, Paul Ingravallo b,1 , Claudia Corredor-Medina a , Basima Al-Khedery a,2 , Marinette Povoa c , John W. Barnwell d a Emory Uniersity, Department of Medicine, Emory Vaccine Research Center, Yerkes Primate Research Center, 954 Gatewood Road, Atlanta, GA 30329, USA b New York Uniersity School of Medicine, 341 East 25th Street, New York, NY 10010, USA c The Instituto Eandros Chagas, Belem, Para, Brazil d Centers for Disease Control and Preention, Diision of Parasitic Diseases, 4770 Buford Highway NE, Chamblee, GA 30341, USA Received 26 September 2000; received in revised form 1 February 2001; accepted 6 March 2001 Abstract The genes encoding two merozoite surface proteins of Plasmodium iax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. iax gt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. iax DASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75 677 Da, and 3 and 5 RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. iax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. iax genes have putative signal peptides, large central domains that contain 20% alanine residues bound by charged regions, are predicted to form -helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low ( 40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3, Pvmsp-3, and Pvmsp-3. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Plasmodium iax ; Malaria; Merozoite; Vaccine; Surface protein; Coiled-coils www.parasitology-online.com. Abbreiations: aa, amino acids; FP, fusion protein; gDNA, genomic DNA; GSP, gene-specific primer; GPI, glycosylphosphatidylinositol; IFA, immunofluorescence assay; IPTG, isopropyl--D-thiogalactopyranoside; MSP, merozoite surface protein; ORF, open reading frame; nt, nucle- otides; UTR, untranslated region; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  Note: Nucleotide sequence data reported in this paper are available in the EMBL, Genbank™ and DDBJ data bases under the accession numbers AF099662 and AF099663. * Corresponding author. Tel.: +1-404-7277214; fax: +1-404-7278199. E-mail address: galinski@rmy.emory.edu (M.R. Galinski). 1 Present address. Schering-Plough Research Institute, Kenilworth, NJ, USA. 2 Present address. University of Florida, Department of Pathobiology, Gainesville, FL 32611-0880, USA. 0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S0166-6851(01)00267-5