Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum

Abstract

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of a single GlcNAc to the Ser or Thr of nucleocytoplasmic proteins. OGT activity, which may compete with that of kinases, is involved in signaling in animals and plants, and abnormalities in OGT activities have been associated with type 2 diabetes. Here, we show that ogt genes that predict enzymes with characteristic tetratricopeptide repeats and a spindly domain are present in some protists (Giardia, Cryptosporidium, Toxoplasma, and Dictyostelium) but are absent from the majority of protists examined (e.g., Plasmodium, Trypanosoma, Entamoeba, and Trichomonas). Similarly, ogt genes are present in some fungi but are absent from numerous other fungi, suggesting that secondary loss is an important contributor to the evolution of ogt genes. Nucleocytosolic extracts of Giardia and Cryptosporidium show OGT activity, and recombinant Giardia and Cryptosporidium OGTs are active in yeast and bacteria, respectively. These results suggest the possibility that O-GlcNAc modification of nucleocytosolic proteins also has function(s) in simple eukaryotes.

Fig. 1

The OGTs of protists, fungus, and bacterium have fewer N-terminal TPRs than previously characterized OGTs of metazoa and plants. Diagrams of selected OGTs show distribution of TPRs and C-terminal, catalytic “spy” domains. Arrow indicates start of recombinant Giardia OGT expressed in Saccharomyces (see Figure 3). Please see supplementary Figures 1 and 2 for alignment of the catalytic domains of Giardia and Cryptosporidium OGTs with those of the most similar metazoan and plant, respectively.

Fig. 2

Phylogenetic analyses suggest a unique origin of the Giardia (Gl) OGT, while OGTs of Cryptosporidium (Cp), Toxoplasma (Tg), and Dictyostelium (Dd) share a common origin with each other. The phylogenetic tree of selected protist, fungal, plant, metazoan, and bacterial OGTs was constructed using the maximum likelihood method. OGTs of Giardia and Cryptosporidium, which were tested here, are highlighted by shaded circles. Lengths of branches are proportional to differences between sequences, while numbers at nodes refer to bootstrap values for 100 trees. Asterisk indicates unresolved central node in this star-shaped tree, so that it is not possible to determine the ancestry of the Giardia OGT. Metazoa include Caenorhabditis elegans (Ce), Canis familiaris (Cf), Drosophila melanogaster (Dm), Danio rerio (Dr), and Homo sapiens (Hs). Fungi include Aspergillus oryzae (Ao), Neurospora crassa (Nc), Magnaporthea grisea (Mg), and Yarrowia lipolytica (Yl). Plants include Arabidopsis thaliana (Ath), Eustoma grandiflorum (Eg), Hordeum vulgare (Hv), Lycopersicon esculentum (Le), Oryxa sativa (Os), and Petunia hybrida (Ph). Bacteria include Anabaena variabilis (Av), Agrobacterium tumefacience (Atu), Bradyrhizobium japonicum (Bj), Nitrosomonas europaea (Ne), and Rhodopseudomonas palustris (Rp).

Fig. 3

OGT activities of Giardia nucleocytosolic extract and of the recombinant GlOGT expressed in yeast. (A) The OGT activity of the Giardia nucleocytosolic extract with itself showed a K_m of 5.8 μM UDP-GlcNAc and a V_max of 5.1 pgm/min/mg protein. (B) The activity of recombinant GlOGT (catalytic domain only), again with itself, showed a K_m of 0.9 μM UDP- GlcNAc and a V_max of 5.6 pgm/min/mg protein. (C) Transfer of a single, O-linked GlcNAc to recombinant GlOGT was confirmed by performing a reductive β-elimination on the precipitated pellet following the OGT assay. Analysis of the products on a Bio-Gel P2 showed a single GlcNAcitol. The negative control in B and C is the empty vector. In each case, representative data are shown from three experiments performed on separate days.

Fig. 4

β-Hexaminidase release of radiolabeled O-GlcNAc from autoglycosylated GlOGT (Figure 3B and C) and from casein kinase (CSK), which was glycosylated by the recombinant CpOGT (Figure 5B and C). In each case, the vast majority of the radiolabeled GlcNAc was released by β-hexosaminidase, confirming the expected β-O-GlcNAc linkage.

Fig. 5

OGT activities of Cryptosporidium nucleocytosolic extract and of the recombinant CpOGT expressed as a GST-fusion protein in bacteria. (A) The OGT activity of the nucleocytosolic extract with itself showed a K_m of 4.3 μM UDP-GlcNAc and a V_max of 5.8 pgm/min/mg protein. (B) The activity of recombinant full-length CpOGT with casein kinase showed a K_m of 5.4 μM UDP-GlcNAc and a V_max of 6.7 pgm/min/mg protein. (C) Transfer of O-linked GlcNAc to casein kinase was confirmed by performing a reductive β-elimination on the precipitated pellet following the OGT assay. Analysis of the products on a Bio-Gel P2 showed a single GlcNAcitol. The negative control in B and C is GST alone. In each case, representative data are shown from three experiments performed on separate days.