Dolichyl-phosphate-glucose is used to make O-glycans on glycoproteins of Trichomonas vaginalis

Abstract

Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about one-fifth the Dol-P-Man synthase activity of Saccharomyces membranes. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.

FIG. 1.

Dol-P-Glc and Dol-P-Man synthase activities of Trichomonas and Saccharomyces membranes. Error bars represent standard deviations of three experiments. Membranes isolated from Trichomonas (Tv) and Saccharomyces (Sc) were incubated with exogenous Dol-P containing 19 isoprene units and either radiolabeled UDP-Glc or GDP-Man. Radiolabeled glycolipids, which partitioned into the organic fraction, were counted and confirmed by TLC in Fig. 2A. Conditions for these assays were determined by varying the pH and the concentration of Trichomonas membranes (see Fig. S1 in the supplemental material).

FIG. 2.

Characterization of the dolichol portion of Trichomonas Dol-P-hexoses. (A) Dol-P-Glc and Dol-P-Man synthase assays were performed as described for Fig. 1, except that (i) exogenous Dol-P was omitted from some of the reactions and (ii) Saccharomyces strains used for Dol-P-Glc synthase assays overexpressed ScALG5. Radiolabeled Dol-P-Glc and Dol-P-Man from Trichomonas (Tv) and Saccharomyces (Sc) membranes were extracted and analyzed by TLC, where the faster-migrating species contain longer dolichol chains. (B) Reverse-phase TLC of dehydrodolichols made by membranes of Saccharomyces and Trichomonas, which were incubated with radiolabeled isopentenyl pyrophosphate and exogenous farnesyl pyrophosphate (in vitro cis-prenyltransferase assay). (C) Mass spectrum of Dol-P-hexose isolated from cultured Trichomonas, showing a major ion peak ([M-H]⁻) with 16 isoprene units and minor peaks with 15 and 17 isoprene units.

FIG. 3.

Phylogenetic reconstruction using the maximum likelihood method of representative Dol-P-Glc synthases (Alg5s) and Dol-P-Man synthases (Dpm1s). Branch lengths are proportionate to differences between sequences, and numbers at nodes indicate bootstrap values for 100 replicates. TvAlg5A to TvAlg5E, which result from gene duplications in Trichomonas, belong to the Alg5 clade of other protists, fungi, and metazoa. TvAlg5G and TvDPM1 belong to no clade, while eukaryotic Dpm1s are split between two groups with (clade A) and without (clade B) a C-terminal transmembrane helix. In addition to Trichomonas vaginalis (Tv), protists include Cryptosporidium parvum (Cp), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg), Leishmania major (Lm), Trypanosoma cruzi (Tc), Trypanosoma brucei (Tb), Dictyostelium discoideum (Dd), and Giardia lamblia (Gl). Fungi include Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Encephalitozoon cuniculi (Ec), while metazoa include Homo sapiens (Hs) and Drosophila melanogaster (Dm). Arabidopsis thaliana (At) is the single plant.

FIG. 4.

Complementation of Saccharomyces alg5Δ mutant with ALG5 genes of Trichomonas. (A) Dol-P-Glc synthase activities with (black bars) or without (gray bars) exogenous Dol-P19 of wild-type (wt) Saccharomyces, as well as a Saccharomyces alg5Δ mutant transformed with the ScALG5 gene and the TvALG5A to TvALG5E, TvALG5G, and TvDPM1 genes. The Dol-P-Glc activity of wild-type Saccharomyces is difficult to detect, as is the Dol-P-Glc synthase activity of the Saccharomyces alg5Δ mutant transformed with TvALG5G. (B) N glycosylation of carboxypeptidase Y (CPY) by the same set of transformants. In Saccharomyces, Dol-P-Glc is used to add three Glc residues to the N-glycan precursor, which is transferred to the nascent peptide. In the absence of Dol-P-Glc synthase activity (TvALG5G and empty vector), the N-glycan precursor lacking Glc is less efficiently transferred to glycoproteins, so CPY, which is detected with an anti-CPY monoclonal antibody, is underglycosylated and runs more quickly on SDS-polyacrylamide gel electrophoresis.

FIG. 5.

Use of real time PCR to estimate the relative abundances of ALG5 and DPM1 mRNAs in cultured Trichomonas (see Table S1 in the supplemental material for the list of primers). A log scale shows that the TvALG5D and TvALG5E mRNAs are much more abundant than those of TvALG5A to -C and TvALG5G, which in turn are much more abundant than that of TvDPM1. Calibrations were relative to a Trichomonas actin gene.

FIG. 6.

Dol-P-Man synthase of TvDpm1 expressed as a recombinant protein in E. coli. E. coli cells were transformed with ScDPM1, TvDPM1, and TvALG5E; recombinant proteins were purified; and their Dol-P-Glc synthase and Dol-P-Man synthase activities were determined by addition of radiolabeled UDP-Glc or GDP-Man, respectively.

FIG. 7.

Characterization of O-glycans attached to endogenous glycoproteins when radiolabeled Dol-P-Glc is added to Trichomonas membranes. Control reactions, which demonstrate the dependence of this reaction on pH, on time, and on temperature, are shown in Fig. S2 in the supplemental material. (A) Radiolabeled glycans released by β-elimination from Trichomonas glycoproteins after incubation of membranes with radiolabeled Dol-P-Glc run with the void (v) volume and total (t) volume of a Bio-Gel P-2 column. (B) Monosaccharide analysis of the radiolabeled O-glycans shows that the large glycan running at the void volume is predominantly Glc, while the single sugar running with the total volume is glucitol (the reducing sugar).