The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N -linked high mannose type structures and low levels of O -linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by approximately 14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove approximately 6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intra-cellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level. These results suggest that N -glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.

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Abstract. Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.

Previously, we reported on the properties of a monoclonal antibody, 2B10, which has the same determinant on the human erythrocyte as MSA-1 of Plasmodium falciparum (FCR3 strain); the binding of both ligands to erythrocyte receptors was totally sialic acid dependent. In this work, rabbit anti-2B10 idiotypic antibodies were generated. The anti-idiotypic antibodies recognized both the erythrocyte binding site of 2B10 and the Cterminal region of MSA-1 (amino acids 1047 to 1640); they were able to inhibit 2B10 and MSA-1 binding to erythrocytes and partially prevent P. falciparum merozoites from invading erythrocytes. The utility of 2B10 in the study of the interaction between MSA-1 and human erythrocytes prompted us to determine the nucleotide and deduced amino acid sequences of its V H and V L regions. The data show that the 2B10 V H region is part of the J558 family and is especially homologous to BALB/c anti-nitrophenyl monoclonal antibody 21.1.43; the V L region belongs to the VK1 subgroup and comes from the same genomic locus as (NZB ؋ W)F 1 anti-DNA and C57BL anti-dextran monoclonal antibodies BXW-14 and 42.48.12.2, respectively. Most of the differences among the V H and V L segments are located in CDR1 and-3. The binding site of 2B10 contains both negatively and positively charged amino acid residues. The amino acid sequences of the 2B10 V H region and a region of MSA-1 from the Wellcome strain of P. falciparum (amino acids 1002 to 1115) share 43% similarity, and the amino acid sequences between the 2B10 V L region and another segment of the same MSA-1 (amino acids 1247 to 1394) share 48% similarity. We conclude that the interactions between erythrocyte receptors and their ligands, 2B10 and MSA-1, are related and that the C-terminal region of MSA-1 is the erythrocyte binding domain.

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