Plasmodium falciparum: molecular analysis of a putative protective antigen, the thermostable 96-kDa protein.

A group of three Plasmodium falciparum antigens of distinct pI, migrating with an apparent MW of 96 kDa has been previously identified as a target of protective immunity both in humans and in monkeys (Jouin et al. 1987, Dubois et al. 1987). These antigens are produced during the late stages of asexual intraerythrocytic development. One of these 96-kDa proteins, the 96 tR, has a pI of 5.25, is thermostable, and is released in the culture supernatant (Jouin et al. 1987). We report here the cloning and expression in Escherichia coli of the gene coding for this antigen. Antibodies raised to the recombinant 96 tR immunoprecipitated exclusively the 96 tR, indicating that the other two antigens of 96 kDa are the product(s) of distinct gene(s). Northern and Southern blots as well as DNA sequencing of the gene showed that the 96 tR antigen is identical to proteins identified in other laboratories as the glycophorin binding protein GBP 130 (Perkins 1984, Ravetch et al. 1985) and Ag 78 (Bianco et al. 1987). The 96-kDa antigen is produced at the trophozoite stage and more actively in the schizonts. It is released in the culture supernatant at the time of schizont rupture, together with two minor products, forming a characteristic triplet. This triplet was also detected in immunoblots of merozoites. An approximate quantification on immunoblots indicated that the largest proportion of the protein is found in the culture supernatant, a minor fraction being loosely associated with merozoites. By immunofluorescence and immunoelectron microscopy, intense signals were observed in the erythrocyte cytoplasm. The 50-amino acid repeats were found in all strains examined, the protein showing some size polymorphism. The antigen was detected in the serum of infected monkeys as well as in that of infected humans.

The polymorphic 11.1 locus of Plasmodium falciparum.

Clone pPF11.1 encodes a Plasmodium falciparum antigen expressed during the intraerythrocytic cycle and containing tandem repeats of a 9 amino acid unit. We report here an analysis of the genomic region specific for 11.1, which extends over 30 kb. It contains two blocks of repeats, spanning 13 kb and 9 kb. The restriction map suggests that the locus may result from a gene duplication. The 11.1 region is present in all P. falciparum strains examined so far. Southern analysis of 8 distinct isolates indicates that the locus is highly polymorphic. Thus the pPF11.1 repeats constitute a sensitive and discriminating probe to type P. falciparum strains.

Karyotype comparison between P. chabaudi and P. falciparum: analysis of a P. chabaudi cDNA containing sequences highly repetitive in P. falciparum.

The molecular karyotypes of P. chabaudi and P. falciparum have been compared by pulse field gradient electrophoresis. P. chabaudi has 3 extra chromosomes in the 750-2000 Kb range although the overall number appears to be 14 as is the case for P. falciparum. The chromosomal location of the rRNA genes has been determined for P. chabaudi together with that of a 24 Kd antigen gene. The corresponding cDNA 443 may code for a protein unusually rich in tyrosine and contains sequences highly repetitive in P. falciparum.

Characterisation of P. falciparum antigenic determinants isolated from a genomic expression library by differential antibody screening.

A genomic expression library of P.falciparum has been differentially screened with a number of immune sera. The response of 9 clones to the various sera is presented, together with the DNA sequence encoding the epitopes. All but one clone are extremely A+T rich and unlike the other P.falciparum epitopes described, are not composed of amino acid repeats. One clone, which responds specifically with a protective serum, has been analysed in detail. The epitope is carried on a 160kd antigen which is transcribed from a single gene to give a protein expressed in all of the erythrocytic forms. DNA sequence of this clone reveals it to have more than one open reading frame, only one of which is transcribed in the blood stages. The possible significance of the other open readings frames is discussed.

Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat.

Plasmodium falciparum causes malaria infections in its human host. Its wide distribution in tropical countries is a major world health problem. Before a vaccine can be produced, the identification and characterization of parasite antigens is necessary. This can be achieved by the cloning and subsequent analysis of genes coding for parasite antigens. Recently established cDNA banks allow the expression of cDNA derived from the simian parasite Plasmodium knowlesi and P. falciparum in Escherichia coli. Recombinants encoding parasite antigens have been identified by immunodetection in both banks. Two of them contain repetitive units of 11 (ref. 7) or 12 (ref. 5) amino acids. We describe here the construction of an expression bank made directly from randomly generated fragments of P. falciparum genomic DNA. We detect several clones which react strongly with human African immune sera. One clone expresses an antigenic determinant composed of occasionally degenerated repeats of a peptide nonamer.

Cloning of cDNAS that code for Plasmodium chabaudi antigens of the erythrocytic forms and cross-hybridize with P. falciparum.

Several Plasmodium chabaudi antigens are synthesized and the corresponding mRNAs are detected by cell-free translation only during specific stages of the erythrocytic cycle. Ring stage mRNA was used to construct a cDNA library in the plasmid vector pBR 322. The library was screened by differential hybridization with cDNA specific for ring stage parasites or schizonts. Two clones which showed some stage specificity and seven which did not were retained. Three clones were characterized by hybrid-selected translation. Clones 451, 148 and 443 contain sequences homologous to the messages for a 27 kDa stage-specific antigen, a 37 kDa early antigen and a 24 kDa antigen, respectively. The nine clones cross-hybridize to various degrees with cDNA from P. falciparum but cross hybridization intensities do not reflect the strength of immunological cross-reactivity.

Two regions of the bifunctional protein aspartokinase I- homoserine dehydrogenase I are connected by a short hinge.

Four proteases differing in their specificity, i.e. subtilisin, trypsin, alpha-chymotrypsin and V8 staphylococcal protease, cleave the bifunctional protein Escherichia coli aspartokinase I-homoserine dehydrogenase I (composed of 820 residues) producing an active homoserine dehydrogenase fragment. This cleavage occurs within a short segment of the polypeptide chain extending from residue 293 to residue 300.

Mechanism of allosteric activation of glycogen phosphorylase probed by the reactivity of essential arginine residues. Identification of an arginine residue involved in the binding of glucose 1-phosphate.

We have previously reported the physicochemical and kinetic properties of glycogen phosphorylase modified by arginine-specific reagent under different conditions [Dreyfus, M., Vandenbunder, B., & Buc, H. (1980) Biochemistry 19, 3634-3642]. The properties of the modified enzyme depend upon the conformation adopted by the enzyme during the modification reaction. In this paper, we report the localization of the crucial modified arginine residues on the primary structure. The chymotryptic peptide extending from residue Asp-563 to residue Tyr-572 was shown to contain one arginine residue (Arg-568) which is chemically modified by phenylglyoxal in phosphorylase a and in activated phosphorlase b. Inclusion of glucose 1-phosphate in the modification medium protects this residue from modification, with a concomitant protection of the enzyme activity. Furthermore, this residue is not reactive toward phenylglyoxal in phosphorylase b in the absence of any effector. Addition of the AMP analogue 2'dAMP, which is not an activator of the enzyme, does not increase Arg-568 reactivity but protects from modification several arginine residues located between Arg-242 and Leu-348. The location and the role of Arg-568 in phosphorylase are discussed with reference to recent data from X-ray crystallography.

Nucleotide sequence of the thrA gene of Escherichia coli.

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.

The primary structure of Escherichia coli K12 aspartokinase I-homoserine dehydrogenase I. Site of limited proteolytic cleavage by subtilisin.

The sequence of the first 25 residues of the homoserine dehydrogenase fragment, produced by limited proteolysis of aspartokinase I-homoserine dehydrogenase I with substilisin, has been determined. The sequence of a cyanogen bromide peptide (CB5, 59 residues), isolated from the entire protein, is also presented. Residues 1 to 18 of the subtilisin homoserine dehydrogenase fragment match the sequence 42 to 59 of peptide CB5.