Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum

Abstract

About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.

FIG. 1.

Proposed biosynthetic pathway of GPI in the ER of P. falciparum. The diagram is reproduced and modified with kind permission from Kinoshita and Inoue (34). In step 1, N-acetylglucosamine is added to PI, and the following steps involve deacetylation, lipidation of the inositol ring, and addition of mannoses. An EtNP on the third mannose enables the linkage of the completed GPI anchor to protein. There is some evidence that steps 8 and 9 can occur in the reverse order. Gene names are given in italics. (Reprinted from reference with permission of the publisher.)

FIG. 2.

Detection of proteins in blood stage parasites was performed using indirect fluorescent-antibody tests. Thin films of parasites at mature stages were fixed in cold acetone and incubated sequentially with 1/80 dilutions of sera from peptide-immunized mice plus rabbit anti-ERC1 (1:200) to determine localization to the parasite ER. These slides were washed and incubated with an appropriate mixture of fluorescein-conjugated anti-mouse and rhodamine-conjugated anti-rabbit (1:1.000) antibodies. Preimmune sera served as negative controls (data not shown). Slides were photographed under appropriate illumination for fluorescein isothiocyanate (first column, showing PIG-A, PIG-O, or DPM1 localization) and rhodamine (second column, showing ERC1 localization), and photographs were overlaid (third column).

FIG. 3.

Best-conserved sequence blocks of PIG-A (A), GPI1 (B), and PIG-C (C). The top lines represent the consensus sequence (CS), which was defined when a residue occurred in at least 51% of the aligned sequences; otherwise, positions are indicated with asterisks. The species are H. sapiens (Hs), S. cerevisiae (Sc), S. pombe (Sp), A. thaliana (At), and P. falciparum (Pf). Dots in the alignments indicate the same amino acid as shown in the consensus; gaps are shown by dashes. The figure was prepared using ASAD (Keith Satterley, ftp://ftp.wehi.edu.au/pub/biology). (A) The best-conserved region in PIG-A. Of the 43 positions, 23 are invariant in the five species. Note that the sequence in P. falciparum adheres to the consensus to a similar degree as the other sequences. The protein from Arabidopsis has an insertion of three amino acids. Accession numbers are NP_002632 (Hs), NP_015150 (Sc), CAB09127 (Sp), and AAK62657 (At). (B) A conserved region of GPI1. These genes evolved faster, but a consensus can be defined for most positions in the conserved blocks. The sequence from P. falciparum again fits the consensus as expected. Some of the variable positions have retained the same character, for example, hydrophobicity. Accession numbers are AAC32661 (Hs), CAB10806 (Sp), AAB17870 (Sc), and NP_191276 (At). (C) A putative fragment of PIG-C in Plasmodium aligned with human and yeast PIG-C sequences. PIG-C is a short and not very strongly conserved protein. We have tentatively identified fragments of the possible homologs in P. falciparum and P. vivax (Pv), essentially based on the alignment shown. The two plasmodial sequences are almost identical over the 30 amino acids. Sources are the GSS sequence AZ573855 (Pv), the truncated cDNA AU087281 (Pf), and the protein submissions Q92535 (Hs) and AAB68262 (Sc).

FIG. 4.

The identification of PIG-L in plasmodia is based on an expressed sequence tag sequence (BF295271) from P. berghei (Pb) and a genomic fragment from P. yoelii (Py). Their sequences are aligned to the PIG-L proteins of H. sapiens (Hs) and S. cerevisiae (Sc), with accession numbers BAA74775 (Hs) and BAA74776 (Sc). The numbers indicate the length of a poorly aligned region in the middle which is not shown. CS, consensus sequence. Symbols and figure preparation are as described in the legend to Fig. 3.

FIG. 5.

Two well-conserved sequence blocks characteristic of the α-1,2-mannosyltransferase PIG-B. The top line represents the consensus sequence (CS). The species are A. thaliana (At), H. sapiens (Hs), S. cerevisiae (Sc), S. pombe (Sp), T. brucei (Tb), Zymomonas mobilis (Z), and P. falciparum (Pf). The Zymomonas protein is a bacterial sequence homolog of unproven function. It serves to tentatively identify the best-conserved positions, which are more likely to be essential for function. (A) The best-conserved sequence block in PIG-B follows the first transmembrane domain and is one of the few hydrophilic segments. Of the 40 positions, only 8 are invariant (12 without Zymomonas), but the consensus can be defined for most positions (27). The bottom line shows that a homologous block of sequence is also present in the Smp3 glycosyltransferase of yeast, with most residues similar and 11 identical to the consensus sequence. (B) The second well-conserved block of PIG-B and Smp3 is also present in other α-1,2-mannosyltransferases, for example, in yeast ALG9 (accession number NP_014180). Accession numbers for PIG-B are CAC01884 (At), BAA07709 (Hs), CAA96854 (Sc), CAB53078 (Sp), BAA94863 (Tb), and AAD53921 (Z); that for yeast Smp3 is NP_014792. Symbols and figure preparation are as described in the legend to Fig. 3.

FIG. 6.

Conserved sequence blocks of the α-1,4-mannosyltransferase PIG-M. A. The first well-conserved block of PIG-M is one of the few hydrophilic regions and closely follows the first transmembrane domains (like in PIG-B). The sequence aligns very poorly with that of the α-1,2-mannosyltransferases. It is strongly conserved among the orthologs, with 15 invariant positions in 36 amino acids (20 when the Trypanosoma sequence, which deviates most from the consensus, is omitted). The D[I,V]D motif (boxed) was identified as essential for activity. (B and C) Two more blocks are strongly conserved, suggesting a tight requirement for a function that remains to be identified. The species and accession numbers are as follows: A. thaliana (At), CAC34506; H. sapiens (Hs), BAB18567; S. cerevisiae (Sc), CAB89880; T. brucei (Tb), BAB20994; and P. falciparum (Pf). For Saccharomyces the nucleotide entry Z49907 was used, as the corresponding protein entries such as CAB89880 are probably based on the wrong start codon. CS, consensus sequence. Symbols and figure preparation are as described in the legend to Fig. 3.

FIG. 7.

Sequence motifs in the GPI phosphoesterases. The species used are H. sapiens (Hs), S. cerevisiae (Sc), S. pombe (Sp), and P. falciparum (Pf). The sequences of yeast Gpi7 (CAA89353) and MCD4 (NP_012756) were added to compare regions that are common to all GPI phosphoesterases. MCD4 aligns poorly with the first block and is omitted there. (A) A region of strong homology between the phosphoesterases of the PIG-O family. The box highlights a PTX[ST]X₈TGX₂P motif present in many alkaline phosphatases. The plasmodial sequence deviates more from the consensus, but most of its substitutions are conservative. The number 5 represents an insertion of five residues in Gpi7. (B) The second block includes the boxed decamer motif HXLGXDHXGH that is also present in many peroxidases. (C) The third block is specific for the GPI phosphoesterases (see text) and may contact the GPI substrate. Accession numbers are AAC07985 (Hs), BAA21454 (Sp), and NP_013069 (Sc). CS, consensus sequence. Symbols and figure preparation are as described in the legend to Fig. 3.

FIG. 8.

(A and B) Alignment of the two best-conserved blocks characteristic of GAA-1. The protein evolved considerable divergence at the amino acid level, and only seven positions are invariant in the two aligned blocks (taken together) with the species A. thaliana (At), H. sapiens (Hs), S. cerevisiae (Sc), and P. falciparum (Pf). Accession numbers are CAA55944 (Sc), NP_197414 (At), and AAH03171 (Hs). (C and D). The histidine (C) and cysteine (D) in the boxed motifs of GPI8 are essential for the transamidation reaction (see text) in other species and are conserved in P. falciparum. Accession numbers are AAB81597 (Hs), P49018 (Sc), T40853 (S. pombe [Sp]), and AJ401202 (Pf). CS, consensus sequence. Symbols and figure preparation are as described in the legend to Fig. 3.

FIG. 9.

Global alignment of the DPM1 protein sequences from A. thaliana (At), H. sapiens (Hs), and P. falciparum (Pf). The alignment shows a high degree of overall conservation. The only gaps are a short deletion in the Arabidopsis gene near the presumptive start codon and a one-amino-acid deletion at position 24 in the Plasmodium protein. The second aspartate in the boxed IDDGS motif and the first aspartate in the boxed MDAD motif are seen in a range of β-glycosyltransferases that transfer a single sugar unit and that have been suggested to participate in catalysis (53). Also boxed is a conserved GTRY motif, where the R is invariant in a broad family that includes sequences of unknown function annotated as DPM1-like proteins in databases. Accession numbers are AAH07073 (Hs) and NP_173481 (At). CS, consensus sequence. Symbols and figure preparation are as described in the legend to Fig. 3.