Ji-ang Li

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCα and PfGCβ) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCβ inEscherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCα was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum.

A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two alpha-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum.

A putative protein serine/threonine phosphatase from Plasmodium falciparum contains a large N-terminal extension and five unique inserts in the catalytic domain.

Plasmodium falciparum possesses a complex life cycle involving two different hosts and interactions with multiple cell types. During the various stages of the life cycle the parasite undergoes cell growth and division, development and differentiation. Sexual stage development, gametocytogenesis in the host and gametogenesis in the mosquito, is accompanied by biochemical and morphological changes in the parasite. The molecular and cellular mechanisms involved in regulation of proliferation, development and differentiation of P. falciparum are unclear. However, inhibitors of protein kinases and phosphatases can interfere with parasite growth [1], suggesting a requirement for phosphorylation–dephosphorylation in control of the parasite life cycle. Reversible phosphorylation has been shown to play an important role in invasion of erythrocytes by merozoites [2] and in intraerythrocytic growth and development [3]. Recently, a number of protein serine/threonine kinase genes have been isolated from P. falciparum [4–7]. Abbre6iations: PPP, PP-1/PP-2A/PP-2B protein serine/ threonine phosphatase family; PfPP-a, Plasmodium falciparum protein serine/threonine phosphatase a ; PfPP-b, Plasmodium falciparum protein serine/threonine phosphatase b ; PFGE, pulse-field gel electrophoresis. * Corresponding author. Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 1865 275722; fax: +44 1865 275721; e-mail: jlli@bioch.ox.ac.uk 1 Note: Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDJB databases under the accession number U88869.

Isolation and characterisation of a cAMP-dependent protein kinase catalytic subunit gene from Plasmodium falciparum☆

The cAMP-dependent protein kinase (PKA) is a key element of the signal transduction pathway by means of the second messenger cAMP. cAMP, generated as a result of activation of membranebound adenylyl cyclases by G protein-coupled surface receptors, exerts nearly all of its effects by activation of PKA [1]. In most organisms, PKA is a heterotetramer consisting of two catalytic and two regulatory subunits [1]. In Dictyostelium, however, PKA is a heterodimer composed of a catalytic and a regulatory subunit [2]. Kinase activity of the catalytic subunit is inhibited by the regulatory subunit. Binding of cAMP to the regulatory subunit alters its affinity for the catalytic subunit, and under physiological conditions, the catalytic subunit dissociates from the regulatory subunit and subsequently phosphorylates many substrate proteins. In mammalian cells, there are three isoforms of the catalytic subunits (Ca, Cb and Cg) and four isoforms of the regulatory subunits (RIa, RIb, RIIa and RIIb) [3] and the tissue-specific expression and assembly of these kinase isoforms are postulated to result in the diverse cellular responses to cAMP [1]. Apart from the regulatory subunits, the heat-stable protein kinase inhibitors (PKIs) are also able to bind the catalytic subunit with a high affinity and subsequently inhibit the kinase activity [1]. Plasmodium falciparum has a complex life cycle involving two different hosts and interactions with multiple cell types. The molecular and cellular mechanisms involved in regulation of proliferation and development of P. falciparum are unAbbre6iations: PKA, cAMP-dependent protein kinase; PfPKAc, Plasmodium falciparum cAMP-dependent protein kinase catalytic subunit; PKIs, the heat-stable protein kinase inhibitors; PfPP-a, Plasmodium falciparum protein serine/ threonine phosphatase a; PfPP-b, Plasmodium falciparum protein serine/threonine phosphatase b; PFGE, pulse-field gel electrophoresis. Note: Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDJB databases under the accession number AF126719. * Corresponding author. Present address: Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK. Tel.: +44-1865-222419; fax: +44-1865-222431. E-mail address: lij@icrf.icnet.uk (J.-L. Li).

Primary structure and sexual stage-specific expression of a LAMMER protein kinase of Plasmodium falciparum

We have isolated a LAMMER-like gene from Plasmodium falciparum by vectorette technique. The gene consists of 3316 bp encoding a protein 881 amino acids with a predicted molecular mass of approximately 106.7 kDa. The encoded protein, termed PfLAMMER, is composed of two distinct domains. The N-terminal domain is not related to any previously described protein kinases and has several interesting features including multiple consensus phosphorylation sites for a range of protein kinases, a number of RS/SR dipeptides, a large proportion of charged amino acids, two putative nuclear localisation signals and 14 copies of a tetramer DKYD repeats. The C-terminal domain is characteristic of a kinase in the LAMMER family with the highest homology to the Arabidopsis thaliana AFC3 kinase. Genomic restriction analysis showed that PfLAMMER is encoded by a single copy gene in the parasite genome. A single transcript of approximately 3800 nucleotides is expressed specifically in the sexual stage, indicating that PfLAMMER may be important in regulating the processes of sexual differentiation of the parasite.

Characterisation of a sexual stage-specific gene encoding ORC1 homologue in the human malaria parasite Plasmodium falciparum.

The origin recognition complex (ORC) is a multisubunit protein composed of six polypeptides that binds to replication origins and is essential for the initiation of chromosomal DNA replication. Using the Vectorette technique, we have isolated a novel gene encoding an ORC1-like protein from the human malaria parasite Plasmodium falciparum. The gene has no introns and encodes a protein (PfORC1) of 1189 amino acid residues with a predicted molecular mass of 139 kDa. PfORC1 contains all conserved sequences in the ORC1/Cdc6/Cdc18 family and displays the highest homology to the Schizosaccharomyces pombe ORC1. However, PfORC1 possesses an extensive N-terminal segment with several interesting features including multiple potential phosphorylation sites, a large proportion of charged amino acids, four copies of a heptamer repeat, two nuclear localisation signals, and a leucine zipper motif. Southern blot analyses show that the Pforc1 gene is present as a single copy per haploid genome and is located on chromosome 12. A 5600 nucleotide transcript of this gene is expressed predominantly in the sexual erythrocytic stage, indicating that PfORC1 may be involved in gametogenesis during which DNA is quickly replicated.

Identification of an MCM4 homologue expressed specifically in the sexual stage of Plasmodium falciparum

Mini-chromosome maintenance (MCM) proteins play an essential role in DNA replication initiation. We have isolated a novel gene encoding an MCM-like protein from the human malaria parasite Plasmodium falciparum using the vectorette technique. The gene has no introns and comprises an open reading frame encoding 1005 amino acid residues with a predicted Mr of 115 kDa. The encoded protein, termed PfMCM4, contains all conserved sequences in the MCM family and displays the highest homology to the Cdc54 (MCM4) of Saccharomyces cerevisiae. However, PfMCM4 possesses five unique amino acid inserts with sizes ranging from seven to 75 residues. Southern blotting of genomic DNA digests and chromosomal separations showed that the Pfmcm4 gene is present as a single copy per haploid genome and is located on chromosome 13. A 4000-nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfMCM4 may be involved in gametogenesis in which DNA is quickly replicated.

Identification of a second proliferating cell nuclear antigen in the human malarial pathogen Plasmodium falciparum

Proliferating cell nuclear antigen seems to exist as a single form in higher eukaryotic cells and plays multiple roles in nucleic acid metabolism. We have identified a second additional proliferating cell nuclear antigen (PfPCNA2) in Plasmodium falciparum on the basis of several lines of evidence. (1) PfPCNA2, consisting of 264 amino acid residues with a predicted molecular mass of 30.2kDa, shares only 29% identity and 53% similarity with PfPCNA1 at the amino acid level. (2) Southern blot analyses revealed that the hybridisation pattern of the Pfpcna2 gene is completely different from that of the Pfpcna1 gene. (3) Chromosomal localisation studies showed that Pfpcna2 is located on chromosome 12 while Pfpcna1 is located on chromosome 13. Northern blot analyses revealed two different transcripts of Pfpcna2, one expressed in both asexual and sexual erythrocytic stages, while the other existed only in the sexual stage, implying that PfPCNA2 may play multiple roles in DNA metabolism in different stages of the parasite. Recombinant protein of PfPCNA2, overexpressed in Escherichia coli, has been purified to near homogeneity and shown to form an oligomer, probably a trimer, as revealed by a size-exclusion chromatography and a native gel electrophoresis, suggesting that PfPCNA2, like its higher eukaryotic counterparts, may serve as a sliding platform which is capable of interaction with diverse proteins and regulation of their activities.

A Family of PP2 Phosphatases in Plasmodium falciparum and Parasitic Protozoa: Reply

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