The nucleotide-sugar transporter family: a phylogenetic approach
Introduction
Two types of donor substrates are used by glycosyltransferases: nucleotide sugars and Dol-P-monosaccharides. The specific transporters of Dol-P-Man and Dol-P-Glc from the cytosol into the endoplasmic reticulum (ER) have not yet been identified [1], it has been proposed to be performed by the corresponding mannosyltransferases and glucosyltransferases, which are multispan, type III proteins [2]. In the case of nucleotide sugars, the transport is performed by a group of eukaryotic transporters, termed nucleotide sugar transporters (NST); due to this role, NST should be considered as partners of the glycosyltransferases in the biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates. This concept is supported by the fact that human pathological states are caused by NST dysfunction.
Congenital disorders of glycosylation (CDG) are characterized by defective N-glycosylation. They comprise two types: (i) the CDG type I are caused by assembly defects of the dolichylpyrophosphate-linked oligosaccharide N-glycan precursor and/or its transfer to asparagines on the nascent polypeptide chain. About 30 known genes are involved in this process; (ii) the CDG type II implies alterations in ulterior steps of the processing of N-glycans. One of the type II disorders, the CDG IIc, is originated by inactivating mutations in the Golgi GDP-fucose transporter (GDP-Fuc) [3], [4], [5]. This disorder produces a primary immunodeficiency syndrome known as leukocyte adhesion deficiencyof type II or LAD II, whose clinical manifestations are mental retardation, short stature, facial stigmata, and recurrent bacterial infections with persistently elevated peripheral leukocytes [6]. The disorder is characterized by lack of fucosylated glycoconjugates, including selectin ligands like the Sia-LeX or the Sia-Lea [3], and lack of H and Lewis blood group antigens [7].
Inactivating mutations of the CMP-sialic acid transporter (CMP-Sia) were recently found to be responsible for another type of CDG; type IIe [8] and Martinez-Duncker et al. (in preparation). In addition, certain important virulence factors in infectious diseases like Leishmaniasis are directly related to a specific NST of the parasite [9]. NST are part of the drug/metabolite transporter superfamily that comprises 14 phylogenetic families of multispan proteins containing from 4 to 10 α-helical transmembrane domains (TMD) [10]. Six of the first seven families only include proteins from prokaryotic organisms, with 4 to 10 TMD. Among the families characterized are the small multidrug resistance family (family 1) and the glucose/ribose and L-rhamnose transporters (families 5 and 6). On the other hand families 8–14 only contain proteins from eukaryotic organisms, and none of the 14 families have both eukaryotic and prokaryotic members. Family 8 are functionally uncharacterized Caenorhabditis elegans proteins that contain nine or 10 putative TMD. Family 9 is the triose phosphate translocator family (TPT) which has been functionally characterized in plants, but also includes some uncharacterized yeast and animal homologues. Families 10–13 consist of the NST proteins with nine to 10 putative TMD, and family 14 contains the plant organocation permeases. In general, the members of this drug/metabolite transporter superfamily participate in (i) drug export, (ii) nutrient and metabolite efflux and (iii) compartmental metabolite exchanges.
An evolutionary hypothesis [10] suggests that the ancestors of the 10 TMD proteins of the drug/metabolite transporter superfamily had four TMD, which first acquired a fifth TMD and then by duplication reached the 10 TMD unit. This hypothesis appears to be applicable to the topology of the CMP-Sia transporter [11] and to the GDP-Fuc transporter that have 10 TMD [4].
As we will frequently refer to the TPT family, it should be noted that only plant sequences were considered, because the functional characterization has only been done in plants, and the metabolic role situates them in the chloroplast, an organelle lacking in yeast and animals. We, therefore, excluded the yeast and animal sequences, and we will refer to this family as the TPT plant family. Also the general context of this TPT family must be distinguished from the particular case of the TPT subfamily, which is one of the four subfamilies of the TPT plant family and is sometimes designated in the literature with the same TPT acronym [10].
Our classification of NST is more extensive than the one presented in the drug/metabolite transporter superfamily classification [10] because it takes into account findings concerning the functional characterization of NST which were not available at the time of that classification.
The genetic engineering of the ER and Golgi apparatus NSTs will provide new ways of creating experimental models that could lead to new therapeutic approaches to disease and improve understanding of the role of this transporters in glycosyltransferase kinetics. Starting from the modification of the substrates used by these transporters, interesting modifications have been performed on nucleotide sugars, as has been the case for the in-agarose production of nucleotide sugars [12], and the synthesis of novel sugar nucleotides which can modify the expression of glycosylation [13]. NST are also directly related to the toxic effects of 3’-azidothymidine-5’ monophosphate (AZTMP), a metabolite derived from 3’-azido-3’-deoxythymidine (AZT) one of the primary chemotherapeutic agents used to treat HIV infection. AZTMP is a potent inhibitor of certain NST [14], [15], and the identification of the chemical groups that interact with the transporters may help to improve drug design. Overexpression of human mRNA of the UDP-Gal/GalNAc Golgi transporter is directly related to the metastatic capacity of colon cancer, by its implication in the synthesis of the Thomsen-Friedenreich, and Sia-Lea and Sia-Lex antigens [16].
This work aimed to provide new objective criteria for the investigation of NST, by defining a comprehensive functional-phylogenetic organization of these transporters.
NST are type III transmembrane solute transporters classified as carriers with an obligatory solute/solute antiport, in accordance to the functional-phylogenetic classification system for transmembrane solute transporters [17] approved by the transport panel of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology.
NST have mainly been characterized in the Golgi apparatus. However, NST located in the ER have also been described, as is the case of the Saccharomyces cerevisiae (sequence 64 of Table 1) UDP-N-acetylglucosamine transporter (UDP-GlcNAc) [18] and the Schizosaccharomyces pombe and S. cerevisiae (sequences 46 and 47) UDP-galactose transporters (UDP-Gal) [19], [20]. In addition, ER carrier-mediated transport of some nucleotide sugars like UDP-glucoronic acid (UDP-GlcA) [21] has been experimentally demonstrated. The donor substrates so far detected in the human ER are UDP-GlcA, UDP-GlcNAc, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-xylose (UDP-Xyl) and UDP-glucose (UDP-Glc), suggesting the existence of corresponding ER transporters [22].
The known chromosome locations of human NST genes (Table 2) show that these genes are spread over different chromosomes.
NST are involved in the transport of nucleotide sugars from the cytosol into the Golgi apparatus or ER lumen via an antiport system, this transport is temperature-dependent and in a saturable manner. For the UDP-GlcNAc transporter, it has been characterized as an electro-neutral transport to the lumen of the Golgi apparatus [23]. The cytosolic nucleotide sugar is exchanged for the corresponding lumenal monophosphate nucleotide. Except for CMP-Sia, which is synthesized in the nucleus [24], the rest of nucleotide sugars are synthesized in the cytosol [25]. For the S. cerevisiae Golgi GDP-Man transporter (sequence 78) and the Kluyveromyces lactis Golgi UDP-GlcNAc transporter (sequence 65), there is experimental evidence that the GMP and UMP counter-substrates are produced in this organelle via type II transmembrane enzymes possessing GDPase and UDPase activity [26], converting diphosphate nucleotides to monophosphate nucleotides, which are then used in the antiport mechanism. Nucleotide diphosphatase activity has also been described in mammalian and fungal Golgi membranes [27]. Fig. 1 illustrates the general transport model of NST.
The hydrophobic multispan topology of NST makes them difficult to crystallize and no structural X-ray diffraction study has been published. All that is known to date is based on computational prediction and experimental results involving mutagenesis, NH2 and COOH tagging, chimera production and immunoprecipitation.
The murine CMP-Sia transporter (sequence 4) is the only NST whose transmembrane topology has been fully studied. It has been shown to have 10 TMD, with the NH2 and COOH terminal ends facing the cytosolic side of the Golgi membrane [11]. Until now, all the functionally characterized NST tagged in their COOH and NH2 terminals show them oriented towards the cytosol. This appears to be a general feature and suggests the presence of an even number of TMD.
The formation of homodimers has been reported for the human Golgi GDP-Fuc transporter (sequence 82), the rat Golgi UDP-GalNAc [28] and GDP-Fuc transporters [29], and the yeast Golgi GDP-Man transporter (sequence 78) [30]. The homodimer structure permits the formation of translocation membrane channels, although its presence has not been demonstrated in all NST transporters. There is evidence of another structure involving a hexameric complex in the Leishmania GDP-Man transporter (sequence 72) [9].
Most of the information about NST structure–function comes from data involving chimeras of human UDP-Gal and CMP-Sia transporters, and shows that different TMD are involved in substrate recognition [31], [32]. This is because the chimera made with the two parental molecules could transport both the UDP and CMP substrates [31]. Substrate specificity with respect to the base of the nucleotide-sugar is high, and no transporter has been identified to transport both UDP and GDP bound sugars, as expected from the structural differences between purines and pyrimidines. The involvement of the base in recognition has been clearly demonstrated for the CMP-Sia acid transporter, which discriminates strongly against purine bases [33]. Chiaramonte et al. [34] who studied the interactions both between the base and the transporter and between the sugar and the transporter, observed that in base-transporter interaction, the transporter makes important contacts with the exocyclic groups at C2 and C4 but tolerates modification at C5 and N3. With respect to the transporter-sugar interaction, these authors found that an intact ribose ring is clearly necessary, and that the 2’-ara hydrogen makes an important contact with the transporter. Another implication of their work is that the lack of the UDP-Gal and UDP-GlcNAc transporters to recognize CMP-Sia may be due to differences in their interactions with the base, and more specifically, to the alteration of the exocyclic C4-amino acid group. NST discrimination between purines and pyrimidines is also indirectly demonstrated by the fact that the thymidine analog AZT, affects the transport of several pyrimidine nucleotide sugars through its metabolic product AZTMP, but does not modify the transport of purine nucleotide sugars [35].
In this study, we included proteins of each of the characterized subfamilies of the TPT plant family, in order to show the close structural and functional relations between the NST families and the TPT plant family.
The TPT plant family proteins differing from NST consist of a mature and a transit peptide of varying length, located in the N-terminus. This transit peptide is enriched in serine, threonine, alanine and valine [36] and its net positive charge is thought to direct it electrostatically to the negatively charged chloroplast envelope, where once inserted, it is cleaved by specific signals [37]. In the case of the TPT subfamily, the cleaving signal motif is P[CP]x↓A, and after cleavage, it leaves the mature peptide. All the functionally characterized plant translocators are located in the inner envelope membranes of chloroplasts and non-green plastids (e.g. amyloplasts, leucoplasts, or chromoplasts) which import carbon as a source of biosynthetic pathways and energy. By experimental and computational methods, these translocators have been structurally described as homodimers [38], [39] allowing the formation of hydrophilic substrate translocation channels in the inner chloroplast membrane [40]. Four subfamilies of the TPT plant family have been identified, with different expression patterns: (i) the TPT subfamily is predominantly present in tissues that perform photosynthetic carbon metabolism; (ii) the phosphoenolpyruvate/phosphate translocator (PPT) subfamily which seems to be ubiquitously expressed; (iii) the glucose-6-phosphate/phosphate translocator (GPT) subfamily, mainly restricted to heterotrophic tissues [41] and (iv) the xylulose-5-phosphate translocator (XTP) subfamily, thought to be expressed in all plant tissues [42].
The TPT subfamily exports fixed carbon, i.e. triose phosphates and the 3-phosphoglycerate derived from C3 photosynthesis, in exchange for inorganic phosphate. In the cytosol, the fixed carbon is used in the formation of sucrose [43]. In the case of C4 mesophyll transporters they also recognize phosphoenolpyruvate (PEP), the substrate for CO2fixation, which contributes to the function of C4 photosynthesis. PEP recognition in C4 plants has appeared after a single mutational event [44].
The PPT subfamily imports PEP into plastids [45], which use it to synthesize aromatic amino acids and related compounds via the Shikimate pathway leading to a series of secondary products.
The GPT subfamily mediates the exchange of glucose-6-phosphate, mainly for inorganic phosphate and triose phosphates [46]. However, in some amyloplasts like the one in wheat endosperm, glucose-1-phosphate rather than glucose-6-phosphate is the precursor for starch biosynthesis. The glucose-6-phosphate of non-green plastids is either used to synthesize starch and fatty acids, or it is fed into the plastidic oxidative pentose phosphate pathway (OPPP) [46].
The XPT subfamily mediates the transport of xylulose-5-phosphate, which is exchanged for inorganic phosphate or triose-phosphate, and to a lesser extent ribulose-5-phosphate, but do not accept ribose-5-phosphate or hexose-phosphate as substrates [42]. The imported substrate is then fed into the OPPP or the Calvin Cycle depending on the plastid where it is located. The XPT proteins share 35–40% of sequence identity with both TPT and PPT sequences, and a higher identity of 50% with GPT sequences. A hypothetical plastid is depicted in Fig. 2 with the four types of TPT family translocator.
The NST and TPT families have common structural and transport mechanisms: both are type III multi-span proteins with about 10 predicted TMD, transport carbohydrates bound to phosphate molecules, are obligatory solute/solute antiporters and form homodimer structures, thus implying that the two families have common properties.
Section snippets
Methods
Seven putative new NST sequences were found by BLAST search of the EST data bank with known NST sequences, followed by multi-alignment of the EST contigs with CAP [47]. The novel NST found by this strategy were annotated and submitted to EMBL for this study. As shown in Table 1, they consist of sequences 12 (Bos taurus AJ440723), 22 (Sus scrofa AJ489473), 38 (B. taurus AJ489254), 40 (Xenopus laevis AJ506039), 50 (X. laevis AJ489472), 83 (S. scrofa AJ440724) and 84 (Mus musculus AJ440720).
We
Phylogeny of NST
A global tree involving the pool of 87 NST and 16 TPT sequences was constructed by neighbor joining with 173 conserved sites after global gap removal. Three main clades were obtained. The first two were named NST family 1 (30 sequences) and NST family 2 (35 sequences). The third consisted of 22 NST sequences defined as NST family 3 and the 16 TPT sequences. Since the bootstrap values for the three clades were significant, each clade was reconstructed separately using the same phylogenetic
Note added in proof
An updated classification of NST that follows the model presented in this article is available at http://www.vjf.cnrs.fr/ial/Fr/laboratoires/U504/inserm_u_504.htm.
Acknowledgements
The present research was partially supported by: Institut National de la Santé et de la Recherche Médicale (INSERM); Centre National de la Recherche Scientifique (CNRS); Association for Research on Cancer (ARC) grant 5348; the French Recombinant Glycosyltransferases Network (G3); the French INSERM/AFM network 4MR29F for Congenital Disorders of Glycosylation (CDG). We thank Dr. Ulf Ingo Flügge for valuable information regarding TPT transport mechanisms, Dr. Kawakita for sharing experimental
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