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 glycosylphosphatidy- linositol (GPI) anchors. Here we show that cultured Giardia makes an unsaturated polyprenyl pyrophosphate (dehydro-dolichol), 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 ofother protists, metazoans and fungi. In transformed Saccharomyces,theGiardia cis-prenyltransferase also makes a polyprenol containing 11 and 12 isoprene units and supports normal growth, N-glycosylation and GPI anchor synthesis of a rer2δ, srt1δ 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. © The Author 2010. Published by Oxford University Press. All rights reserved.