The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases

Abstract

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.

Fig. 1.

The inventory of Alg glycosyltransferases and predicted dolichol-linked glycans vary dramatically among protists and fungi. Predicted Alg glycosyltransferases and dolichol-linked glycans of Saccharomyces cerevisiae, Homo sapiens, and Dictyostelium discoideum (A), Trypanosoma cruzi, Trypanosoma brucei, Leishmania major, and Cryptococcus neoformans (B), Tetrahymena thermophilia, Toxoplasma gondii, and Cryptosporidium parvum (C), Entamoeba histolytica and Trichomonas vaginalis (D), Plasmodium falciparum and Giardia lamblia (E), and Encephalitozoon cuniculi (F) (see also Table 1). With the exceptions of Saccharomyces and Homo, sets of Alg glycosyltransferases were identified here. Names of organisms, whose dolichol-PP-linked glycans were previously identified (e.g., Saccharomyces), are indicated in black. Names of organisms, whose dolichol-PP-linked glycans were identified here (e.g., Cryptococcus), are indicated in red. Names of organisms, whose dolichol-PP-linked glycans have not yet been identified (e.g., Cryptosporidium), are indicated in green.

Fig. 2.

Common ancestry is a useful method of predicting the Alg glycotransferase inventory of each eukaryote. Phylogenetic reconstructions by using the maximum likelihood method of representative eukaryotic and prokaryotic Alg7 (A) and Alg1, Alg2, and Alg11 (B). Branch lengths are proportionate to differences between sequences, and numbers at nodes indicate bootstrap values for 100 replicates. Eukaryotes include Arabidopsis thaliana (At), Cryptococcus neoformans (Cn), Cryptosporidium parvum (Cp), Dictyostelium discoideum (Dd), Entamoeba histolytica (Eh), Giardia lamblia (Gl), Homo sapiens (Hs), Leishmania major (Lm), Plasmodium falciparum (Pf), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Tetrahymena thermophilia (Tt), Toxoplasma gondii (Tg), Trichomonas vaginalis (Tv), Trypanosoma brucei (Tb), and Trypanosoma cruzi (Tc). Archaea include Euryarchaeota Archaeoglobus fulgidus (Af), Ferroplasma acidarmanus (Fa), Methanococcoides burtonii (Mb), Methanococcus jannaschii (Mj), Methanopyrus kandleri (Mk), Methanosarcina mazei (Mm), Picrophilus torridus (Pt), Pyrococcus abyssi (Pab), Pyrococcus furiosus (Pfu), Pyrococcus horikoshii (Ph), and Crenarchaeota Pyrobaculum aerophilum (Pae), Sulfolobus solfataricus (Ss), and Sulfolobus tokodaii (St). Bacteria include Actinobacillus actinomycetemcomitans (Aa), Bifidobacterium longum (Bl), Borrelia garinii (Bg), Burkholderia cepacia (Bc), Clostridium acetobutylicum (Ca), Enterococcus hirae (Ehi), Listeria innocua (Li), Staphylococcus aureus (Sa) Streptococcus pneumoniae (Spn), Synechococcus elongates (Se), Thermus thermophilus (Tth), and Tropheryma whipplei (Tw). Not all organisms are present in each tree.

Fig. 3.

Predicted Trichomonas, Entamoeba, and Cryptococcus dolichol-PP-glycans were identified in vivo and in vitro. Dolichol-PP-linked precursors from Trichomonas vaginalis (A), Entamoeba histolytica (B), and Cryptococcus neoformans (C), as well as in vitro OST assays by using membranes from Trichomonas (D), Entamoeba (E), and Cryptococcus (F). In A-C, glycans were labeled in vivo with [³H]Man and separated on a P-4 column, whereas in D-F, glycans were transferred to a radio-iodinated tripeptide NYT in vitro, captured with Con A, and separated by HPLC.

Fig. 4.

Giardia dolichol- and N-linked glycans are composed of GlcNAc and GlcNAc₂. Dolichol-PP-linked glycans (A) and N-linked glycans (B) of Giardia lamblia, each labeled in vivo with [³H]GlcN and separated on a P-4 column. Solid lines indicate labeled products, whereas dotted lines indicate products of digestion of the excised GlcNAc₂ peak with chitobiase. Giardia dolichol- and N-linked glycans are composed of GlcNAc and GlcNAc₂.

Fig. 5.

The present diversity of protist and fungal dolichol-PP-lined glycans appears to result from secondary loss glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases. Models suggesting sequential addition (A) or secondary loss (B) of Alg glycosyltransferases during eukaryotic evolution are shown. Nodes, which are labeled numerically in A and alphabetically in B, are explained in the text.