Kinetoplastid Metabolism
Kinetoplastid Metabolism
2016
Sugar nucleotides are the activated form of monosaccharides. They function as glycosyl donors in glycosylation reactions. The resulting glycoconjugates, expressed on the surface of trypanosomatid parasites, fulfill a vital role in host-parasite interaction and they are essential for infectivity, virulence and parasite survival inside the host. There exists a considerable body of work, mainly from the laboratory of Michael Ferguson, describing their structure, biosynthesis, and function. A study (Turnock and Ferguson, 2007) of the size of the sugar nucleotide pools and the enzymes involved in their synthesis provides an excellent insight into the pathways of sugar nucleotide synthesis present in the three major trypanosomatid representatives: T. brucei, T. cruzi and L. major.
Turnock and Ferguson concluded that five sugar nucleotides: GDP-alpha-D-mannose, UDP-alpha-D-N-acetylglucosamine, UDP-alpha-D-glucose, UDP-alpha-galactopyranose, and GDP-beta-L-fucose, were common to T. brucei, T. cruzi and L. major; one, UDP-alpha-D-galactofuranose, was common to T. cruzi and L. major; three, UDP-beta-L-rhamnopyranose, UDP-alpha-D-xylose, and UDP-alpha-D-glucuronic acid, were found only in T. cruzi, and one, GDP-alpha-D-arabinopyranose, was found only in L. major.
The recent identification of orthologous genes in nine additional trypanosomatids and one bodonid genome has allowed to extend the study of Turnock and Ferguson to all available Kinetoplastea.
The orthologs for each of the enzymes of sugar nucleotide synthesis for thirteen kinetoplastid organisms are shown in Table 1.
GDP-alpha-D-mannose (GDP-Man, enzymes #1,2,7,8,9, Fig 1) should be formed by all the Kinetoplastea because it is an obligate precursor to the dolichyl-P-Man donor in the synthesis of glycoconjugates present in all eukaryotes. It is formed, either from glucose, or from the gluconeogenic and pentose-phosphate intermediate fructose 6-phosphate (F6P). Although enzyme #2 was not detected in Bodo saltans this essential glycolytic enzyme, or another isofunctional enzyme, must be present. Indeed, isofunctional enzymes of an absent phosphoglucomutase in the African trypanosomes have already been identified ( see below and Bandini et al., 2012) Enzyme #8 is absent in the Phytomonas Hart1 isolate, but present in the EM isolate. Here also an isofunctional enzymes with mutase activity may have taken over this function. Enzyme #9 is absent in T. congolense only. However, since we know that T. congolense makes GPIs and N-glycans, it should be able to make GDP-Man as well. Maybe PGM (#12) and/or PAGM (#5) have isofunctional activities here (M. Ferguson, personal communication).
UDP-alpha-D-N-acetylglucosamine (UDP-GlcNac; enzymes #1,2,3,4,5,6) is abundantly present in the TriTryps. Both Paratrypanosoma and Phytomonas lack enzyme #3, but these organisms most likely convert external GlcN to GlcN6P by exploiting the broad substrate specificity of the enzyme hexokinase. One of the two Phytomonas strains has lost an essential gene (enzyme #6) in the formation of UDP-GlcNAc (Table. 1). Without UDP-GlcNAc Phytomonas would not be able to make chitin, a surface polysaccharide of N-acetylglucosamine and previously identified in Phytomonas françai (Nakamura et al., 1993; Gomes-Rocha et al., 2003), or GPIs or N-links. This is highly unlikely, because it would be the only known eukaryote to lack both. Most likely this job is carried out by another isofunctional pyrophosphorylase (M. Ferguson, personal communication).
UDP-alpha-D-glucose (UDP-Glc, enzymes #1,12,13) is present in the three TriTryps. It is the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum (Izquierdo et al., 2009). It is also needed for the formation of glycosylated β-D-hydroxymethyluracyl, or base J, a trypanosomatid-specific essential DNA modification that prevents readthrough at RNA polymerase II termination sites (Van Luenen et al., 2012). Therefore, it should be essential to all trypanosomatids and probably also to Bodo saltans. Gene #12, phosphoglucomutase, is absent from the three African trypanosomes, but yet the enzymic activity is present (Opperdoes, unpublished). Recently it was reported that phospho-N-acetylglucosamine mutase (PAGM, #5) and also phosphomannomutase (PMM, #8) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of N-acetylglucosamine and also act on glucosephosphates. The existence of these side activities must have facilitated the loss of the phosphoglucomutase gene from the common ancestor of T. brucei and the other African trypanosomes (Bandini et al., 2012). Enzyme #13, UDP-glucose pyrophosphorylase, is absent from T. vivax and the two Phytomonas genomes. How UDP-Glc is formed in these two trypanosomatids is not yet clear, but the presence in these two #13-lacking parasites, of a recently characterised UDP-sugar pyrophosphorylase (USP, #13a), which in L. major is involved in both UDP-gal and UDP-Glc biosynthesis (Damerow et al., 2015) suggests that this enzyme may compensate for the loss of #13 in T. vIvax and Phytomonas as well.
UDP-alpha-galactopyranose (UDP-Galp, enzymes #16,17,14) is formed from UDP-Glc (#14), or by salvage from galactose (#16,17). In Paratrypanosoma, Blechomonas and the African trypanosomes UDP-Gal cannot be formed from galatose because galactokinase is absent while the hexose transporter is unable to transport Gal (Barrett et al., 1998). Thus the only route to UDP-Galp is via the epimerization of UDP-Glc to UDP-Galp (by enzyme #14) (Urbaniak et al., 2006).
UDP-alpha-D-galactofuranose (UDP-Galf) is formed from UDP-GalP by enzyme #15. However, the enzyme is neither present in Paratrypanosoma , Blechomonas, and the African tryps, nor in Phytomonas.
GDP-beta-L-fucose (GDP-Fuc; enzymes #8,9,10,11); This pathway does not operate in Blechomonas and the Leishmaniinae because of the absence the enzymes #10 and #11. Nevertheless Leishmania is known to contain tiny amounts of GDP-Fuc, probably because the enzymes that make GDP-Ara (see below) can also make GDP-Fuc (Turnock and Ferguson, 2007, Novozhilova and Bovin, 2009). One of the two Phytomonas strains (Hart 1) has lost #10 and #11 as well.
GDP-alpha-D-arabinopyranose (GDP-Ara), is found only in L. major and C. fasciculata (Turnock and Ferguson, 2007). Although the pathway of its formation has not yet been elucidated and none of the genes involved in the pathway clearly identified, arabinose and fucose differ by only one methyl group and arabinose kinase (#23) displays also some fucose kinase activity (Turnock and Ferguson, 2007). Interestingly, trypanosmatids lacking the enzymes #10 and #11 (e.g. Blechomonas and all the Leishmaniinae) do have a copy of a fucose kinase / arabinose kinase (#23). The two gene copies found in T. cruzi are probably truncated pseudogenes. Thus only the EM1 and Hart1 strain of Phytomonas seem to lack the possibility to form any arabinose- and fucose-containing glycoconjugates.
UDP-beta-L-rhamnopyranose (UDP-Rha, #18,19) is not formed in the African trypanosomes and in the Phytomonads, because both enzymes, #18 and19, are absent.
UDP-alpha-D-xylose (UDP-Xyl, #20,21) is not formed in Blechomonas, the African trypanosomes and the group of Leishmaniinae, becasue of the absence of the enzymes #20 and 21 in these organisms.
In conclusion, Bodo saltans and T. cruzi seem to be the two most versatile organisms when it comes to the variety of sugars that can be formed for subsequent use in glycosylation reactions. Bodo seems only to be hampered in the formation of GlcNAc sugars, although it is likely that this kinetoplastid obtains its GlcN directly form its prey and /or endosymbiotic bacteria and subsequently phosphorylates it with the enzyme hexokinase (see above). African tryps lack the possibility to incorporate Galf Rha and Xyl. Phytomonads seem to lack the capacity to incorporate GlcNAc, Galf and Rha, but together with Bodo, Paratrypanosoma and T. cruzi should be able to form UDP-GlcUA and UDP-Xyl. Finally, the Leishmaniinae are unable to form, Rha and Xyl-containing glycoconjugates.
Special acknowlegement: The critical comments on this section by Michael Ferguson were higly appreciated.
25. Synthesis of sugar nucleotides
07/10/16
Figure 1. overview of the sugar nucleotide biosynthesis pathways in three trypanosomatid parasites. The sugar nucleotides used for glycoconjugate biosynthesis are in bold. Sugar nucleotide biosynthetic intermediates are in parentheses. Salvage pathways are in italics. The numbers refer to the enzymes and known or candidate genes described in Table 4 from Turnock and Ferguson, 2007 and also mentioned in the Table below. The colors of the arrows and underlining indicate the presence of the corresponding enzyme and sugar nucleotide, respectively, in T. brucei (orange), T. cruzi (green), and L. major (red). Figure taken from Turnock and Ferguson, 2007.
Table 1. Identification of the genes involved in sugar nucleotide formation for all of the Kinetoplastea. Green fields, gene identified; red fields, gene absent. Yellow fields, pseudogene. Click on the table for more details.
alpha-D-glucose
N-acetylglucosamine
Fucose
Rhamnopyranose
Xylulose
Glucuronic acid
By Fred R. Opperoes
E-mail:Fred.Opperdoes@uclouvain.be
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