Molecular characterization of the cis-prenyltransferase of Giardia lamblia

Abstract

Giardia lamblia, the protist that causes diarrhea, makes an Asn-linked-glycan (N-glycan) precursor that contains just two sugars (GlcNAc(2)) attached by a pyrophosphate linkage to a polyprenol lipid. Because the candidate cis-prenyltransferase of Giardia appears to be more similar to bacterial enzymes than to those of most eukaryotes and because Giardia is missing a candidate dolichol kinase (ortholog to Saccharomyces cerevisiae SEC59 gene product), we wondered how Giardia synthesizes dolichol phosphate (Dol-P), which is used to make N-glycans and glycosylphosphatidylinositol (GPI) anchors. Here we show that cultured Giardia makes an unsaturated polyprenyl pyrophosphate (dehydrodolichol), which contains 11 and 12 isoprene units and is reduced to dolichol. The Giardia cis-prenyltransferase that we have named Gl-UPPS because the enzyme primarily synthesizes undecaprenol pyrophosphate is phylogenetically related to those of bacteria and Trypanosoma rather than to those of other protists, metazoans and fungi. In transformed Saccharomyces, the Giardia cis-prenyltransferase also makes a polyprenol containing 11 and 12 isoprene units and supports normal growth, N-glycosylation and GPI anchor synthesis of a rer2Delta, srt1Delta double-deletion mutant. Finally, despite the absence of an ortholog to SEC59, Giardia has cytidine triphosphate-dependent dolichol kinase activity. These results suggest that the synthetic pathway for Dol-P is conserved in Giardia, even if some of the important enzymes are different from those of higher eukaryotes or remain unidentified.

Fig. 1

Mapping of predicted Giardia enzymes onto the eukaryotic synthetic pathway for dolichol phosphate (Dol-P). Orthologs to Saccharomyces enzymes, which are present in Giardia (e.g. Rer2/Srt1, Alg7 and Dpm1) are marked in red. Saccharomyces Sec59, which appears to be absent in Giardia, is marked in orange. Enzymes that have not been molecularly characterized and so are not identified by searches with BLASTP (e.g. Dedol saturase) are marked in gray. Two alternative pathways for Dol-P synthesis in Giardia, which are tested by experimentation here, are the conversion of Dedol-P to Dol-P (marked in green) and use of Dedol-P rather than Dol-P for synthesis of N-glycans and GPI anchors (marked in blue)

Fig. 2

Thin layer chromatography (TLC) shows the Giardia undecaprenyl pyrophosphate synthase (Gl-UPPS), which closely resembles the bacterial UPPS (Supplement Figure 1 and see Figure 4 below) that makes a dominant polyprenol pyrophosphate with 11 isoprene units (Dedol-11). (A) In the left lane, Giardia membranes incubated with radiolabeled IPP make Dedol-11. Faster running label represents short chain prenols. In the right lane, membranes from a Saccharomyces rer2Δ, srt1Δ double-mutant expressing Gl-UPPS also make Dedol-11 when incubated with radiolabeled IPP. (B) Recombinant Gl-UPPS, which was purified from transformed E. coli, makes polyprenol pyrophosphate with 11 isoprene units (Dedol-11-PP), which is converted to Dedol-11-P by treatment with bacA, which is an undecaprenyl pyrophosphate phosphatase. In both A and B, the origin is at the bottom of the TLC plate. In A the resolving zone is shown, while in B the origin is also shown

Fig. 3

Mass spectroscopy of the products of the Giardia cis-prenyltransferase (Gl-UPPS) reveals dolichols containing 11 and 12 isoprene units. (A) Dolichols extracted from Giardia trophozoites growing in axenic culture (without bacteria) include Dol-11 and Dol-12. The absence of phosphate and pyrophosphate groups on the polyprenols implies the existence of endogenous Giardia Dedol-PP pyrophosphatases and Dedol-P phosphatases. (B) Dolichols isolated from wild-type Saccharomyces (BY4741) containing intact RER2 and SRT1 genes contain 14 to 17 isoprene units. (C) Transformed Saccharomyces rer2Δ, srt1Δ double-mutant, which is expressing Gl-UPPS, makes Dol-11 and Dol-12. While the Saccharomyces rer2Δ, srt1Δ double-mutant is dependent upon expression of Gl-UPPS for growth (Figure 5), these cells do not show defects in N-glycan or GPI anchor synthesis, using CPY and Gas1p, respectively, as reporters (Supplemental Figures 2 and 3, respectively)

Fig. 4

The Giardia undecaprenyl pyrophosphate synthase (Gl-UPPS) resembles cis-prenyltransferases of eubacteria, archaea, Trypanosoma and plant chloroplast. In the phylogenetic tree, which was constructed by maximum likelihood methods, the branch lengths are proportionate to differences between sequences, while the nodes indicate bootstrap support. Nodes with less than 50% bootstrap support were collapsed. Representative protists include Giardia (Gl), Cryptosporidium parvum (Cp), Dictyostelium discoideum (Dd), Leishmania major (Lm), Paramecium tetraurelia (Pt), Plasmodium falciparum (Pf), Tetrahymena theromophila (Tt), Trichomonas vaginalis (Tv) and Trypanosoma brucei (Tb). Representative metazoans include Drosophila melanogaster (Dm), Homo sapiens (Hos) and Xenopus laevis (Xl). Representative fungi include Kluyveromyces lactis (Kl), Pichia pastoris (Pp), Saccharomyces cerevisiae (ScRer2 and ScSrt1) and Schizosaccharomyces pombe (Sp). Also included are chloroplast (At1) and cytosolic (At2) cis-prenyltransferases of Arabidopsis thaliana, as well as that of Oryza sativa (Os). Representative eubacteria include Escherichia coli (Ec-PDB) and Micrococcus luteus (Ml), each of which has been crystallized (Fujihashi et al. 2001; Guo et al. 2005), as well as Bacillus subtilis (Bs) and Staphylococcus aureus (Sa). Representative archaea include Pyrococcus furiosus (Pyf) and Halobacterium sp. (Has)

Fig. 5

Functional complementation of Saccharomyces rer2Δ, srt1Δ double-deletion mutant by the Giardia cis-prenyltransferase (Gl-UPPS). Wild-type yeast (BY4741), the rer2Δ deletion strain, the rer2Δ strain expressing Gl-UPPS or the rer2Δ, srt1Δ double-deletion strain expressing Gl-UPPS were streaked onto YPD plates or synthetic complete medium containing 1% 5-fluoroorotic acid (FOA). The Ura3 protein, which is expressed from the URA3 marker present in the plasmids, converts FOA to toxic 5-fluorouracil. Wild-type yeast and the single the rer2Δ deletion strain are each able to grow in the absence of plasmid, while the rer2Δ, srt1Δ double-deletion strain is unable to grow in the absence of the Gl-UPPS expressing plasmid

Fig. 6

Despite the absence of an ortholog to Saccharomyces SEC59, Giardia has a CTP-dependent dolichol kinase. Membranes isolated from Giardia and Saccharomyces were incubated and radiolabeled with [γ³²P]CTP +-//0− exogenous dolichol containing 19 isoprene units. Radiolabeled alkaline-stable phospholipids, which partitioned into the organic fraction, were counted