Cloning and expression of nucleotide sugar or PAPS transporter genes

Nucleotide sugar transporters (NSTs) are multimembrane-spanning proteins in the plane of the endoplasmic reticulum (ER) or Golgi membrane and are crucial for the synthesis of glycoconjugates (Fig. 1) ^2,5). Glycosylation is performed by various types of glycosyltransferase in the lumens of the ER and the Golgi apparatus.  All glycosyltransferases require high-energy donor sugars activated by the addition of a nucleoside mono- or diphosphate (CMP, UDP or GDP), which are referred to as nucleotide sugars. Nucleotide sugars are synthesized in the cytosol (or in the nucleus in the case of CMP-sialic acid). The translocation of nucleotide sugars from the cytosol into the lumen of each compartment is mediated by NSTs. NSTs transport nucleotide sugars by coupling with the antiport of nucleoside monophosphate (NMP), which is produced as the result of a glycosyltransferase reaction and a subsequent luminal nucleoside diphosphatase (NDPase) reaction. Recently, some NSTs have been reported to transport nucleotide sugars by coupling with the antiport of another nucleotide sugar or nucleoside diphosphate (Muaraoka et al., 2007). Indeed, NSTs are likely to be key components in the synthesis of glycoconjugates.

The nucleotide sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation.  Sulfate is transferred from PAPS to a defined position on the sugar residue or proteins by sulfotransferases. In a similar fashion to nucleotide sugars, PAPS is synthesized in the cytosol by a PAPS synthetase and is translocated from the cytosol into the Golgi lumen through a PAPS transporter (PAPST) ^1,3). Thus PAPS transporters belong to the nucleotide sugar transporter family (Fig.2). 

Since the original cloning of NST, which was reported in 1996, many other NSTs have been cloned and their transport activities identified (Table 1). The Hugo Nomenclature committee categorizes NSTs and PAPSTs as solute carrier transporters that do not perform active transport using energy derived from ATP. This group of transporters comprise the SLC35 (solute carrier 35) family of proteins (Table 1). The SLC35 family is divided into five sub-groups, A~F. In addition to these NSTs, many GDP-Man transporters have been identified in plants and fungi (Fig. 2).

Ten human NST genes, including PAPS transporters, have now been cloned and their protein activities identified (Table1, Fig.2). Among them, four NSTs have multi-substrate specificities²⁾. Four other NSTs transport single specific nucleotide sugars, and a further two transport PAPS1,3).  The inactivation of the GDP-Fuc transporter (SLC35C1) and the CMP-Sia transporter (SLC35A1) causes congenital disorders of glycosylation (CGD)-IIc/ leukocyte adhesion deficiency type II and CGDIIf, a new type of CGD-II, respectively (Table1). In model organisms, such as C. elegans, Drosophila, Leishmania, and yeast, mutations of NST genes have been identified and used for the functional analysis of NSTs, which will help to clarify the physiological functions of sugar chains.

1. Kamiyama, S., Suda, T., Ueda, R., Suzuki, M., Okubo, R., Kikuchi, N., Chiba, Y., Goto, S., Toyoda, H., Saigo, K., Watanabe, M., Narimatsu, H., Jigami, Y., and Nishihara, S. (2003) Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter. J Biol Chem. 278, 25958-25963 [PMID : 12716889]
2. Suda, T., Kamiyama, S., Suzuki, M., Kikuchi, N., Nakayama, K., Narimatsu, H., Jigami, Y., Aoki, T., and Nishihara, S. (2004) Molecular cloning and characterization of a human multisubstrate specific nucleotide-sugar transporter homologous to Drosophila fringe connection. J Biol Chem. 279, 26469-26474 [PMID : 15082721]
3. Kamiyama, S., Sasaki, N., Goda, E., Ui-Tei, K., Saigo, K., Narimatsu, H., Jigami, Y., Kannagi, R., Irimura, T., and Nishihara, S. (2006) Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2. J Biol Chem. 281, 10945-10953 [PMID : 16492677]
4. Muraoka, M., Miki, T., Ishida, N., Hara, T., and Kawakita, M. (2007) Variety of nucleotide sugar transporters with respect to the interaction with nucleoside mono- and diphosphates. J Biol Chem. 282, 24615-24622 [PMID : 17599910]
5. Nishihara, S. (2008) Nucleotide sugar transporter genes and their functional analysis. Experimental Glycoscience - Glycobiology, edited by Naoyuki Taniguchi et al., Springer, Part1, Section III, 103-107.