2 Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-501, Japan, and 3 CREST JST, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-501, Japan
Received on August 2, 2004; revised on September 29, 2004; accepted on October 1, 2004
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Abstract |
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Key words: acceptor specificity / glucuronyltransferase / HNK-1 carbohydrate
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Introduction |
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Recently we purified and cloned a GlcAT (GlcAT-P) from rat brain, which is involved in the biosynthesis of the HNK-1 carbohydrate (Terayama et al., 1997, 1998
). Using rat GlcAT-P cDNA, we and others have cloned a second GlcAT (GlcAT-S) (Seiki et al., 1999
; Shimoda et al., 1999
). The transfection of GlcAT-P or GlcAT-S cDNA into COS-1 cells resulted in the expression of the HNK-1 carbohydrate in glycoproteins on the cell surface. Characterization of GlcAT-P purified from rat brain revealed that the expression of the GlcAT activity toward glycoprotein acceptors, for example, asialo-orosomucoid (ASOR), depends almost completely on sphingomyelin (SM). Under these assay conditions (i.e., in the absence of phosphatidylinositol [PI]), however, there was no activity toward a glycolipid acceptor, palagloboside (Terayama et al., 1998
). GlcAT-P specifically recognized the N-acetyllactosamine structure at the nonreducing terminals of glycoprotein acceptors (Oka et al., 2000
; Terayama et al., 1997
, 1998
).
More recently, we succeeded in generating mice with targeted deletion of the GlcAT-P gene (Yamamoto et al., 2002). To our surprise, the GlcAT activity toward paragloboside disappeared almost completely, as well as the activity toward ASOR (Yamamoto et al., 2002
). Thus it would be interesting to determine whether GlcAT-P really exhibits activity toward glycolipid acceptors. Furthermore, the HNK-1 carbohydrate disappeared almost completely in GlcAT-P-deficient mice, but a trace of HNK-1 immunoreactivity remained on the surfaces of the soma and proximal dendrites of a subset of neurons in some limited regions. These remaining HNK-1 carbohydrates in GlcAT-P deficient mice are localized predominantly in the perineuronal nets and are assumed to be synthesized by GlcAT-S (Yamamoto et al., 2002
). These results suggest the possibility that the two glucuronyltransferases synthesize structurally and functionally different HNK-1 carbohydrates in vivo.
In this study, we prepared protein A fusion recombinant GlcAT-P and GlcA-S in COS-1 cells, and then analyzed the acceptor specificities of the two glucuronyltransferases. GlcAT-P was shown to exhibit activity not only toward a glycoprotein acceptor, ASOR, but also toward a glycolipid acceptor, paragloboside, the latter activity requiring the presence of phospholipids such as PI.
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Results |
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The specificity of the activity toward NCAM
Throughout this study, we used ASOR, which is a serum glycoprotein, as a model acceptor substrate to measure the activity toward glycoproteins. To determine the effect of the polypeptide portion of the acceptor substrates, we used rat NCAM, which bears the HNK-1 carbohydrate in vivo. The soluble form of rat NCAM was expressed as an Fc-fusion protein in Lec2 cells, and purified as described under Materials and methods. Lec2 cells are mutant Chinese hamster ovary cells. Glycoproteins and glycolipids produced by these cells lack sialic acid (Stanley and Siminovitch, 1977). As shown in Figure 4, NCAM-Fc prepared from Lec2 cells was detected with RCA120 lectin, which recognizes terminal galactose residues, indicating that the purified NCAM-Fc has terminal galactose residues that are used by GlcAT-P and GlcAT-S. ASOR and NCAM have different numbers of N-linked sugar chains on their peptide portions.
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Discussion |
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Hydropathy analysis revealed that GlcAT-P and GlcAT-S are structurally similar to previously identified GlcATs (Paulson and Colley, 1989) in that they are type II transmembrane proteins comprising a short N-terminal cytoplasmic tail, a transmembrane domain, a stalk, and a large globular catalytic domain (Colley, 1997
). In this study we only used the catalytic domain, a truncated form, of GlcAT-P and GlcAT-S. In the cellular membrane system, SM is located predominantly in the outer leaflet of the plasma membrane and the Golgi apparatus (Abe and Norton, 1974
) and is presumed to interact with the catalytic domain of GlcAT-P. On the other hand, PI is mainly localized in the inner leaflet of the plasma membrane and the Golgi apparatus. A possible explanation may be that there are other similar lipids in the outer leaflet, as such as the GPI portion of GPI anchor proteins acts as PI to regulate the activities of GlcAT-P and GlcAT-S in vivo. Some phospholipids have been reported to enhance the activities of glycosyltransferases, such as hepatic GlcAT (Pukazhenthi et al., 1993
), ß1-4 galactosyltransferase (Yamaguchi and Fukuda, 1995
), and
2-3-sialytransferase (Nilsson and Dallner, 1977
). However, to our knowledge, this is the first report indicating that the activity of a glycosyltransferase is regulated by two membrane lipids, SM and PI, to switch its activity toward a glycoprotein acceptor substrate or a glycolipid acceptor substrate. These lines of evidence suggest that expression of the HNK-1 epitope can be regulated not only by the expression level of the enzyme protein but also by the microenvironment around the enzyme, especially by the presence of SM and PI.
It should be noted that both enzymes showed little activity toward Lewis x or Lewis y trisaccharides (Table III), although both of them have the N-acetyllactosamine structure, indicating that a fucose residue on GlcNAc inhibited the activities of GlcAT-P and GlcAT-S toward the N-acetyllactosamine structure. Andressen et al. (1998) examined the terminal carbohydrate sequences of the lactoseries on the developing chick olfactory receptor epithelium. They found HNK-1 epitope expression only in the immature olfactory receptors and Lewis x epitope expression only in the mature olfactory receptors, although N-acetyllactosamine was expressed on the whole epithelial cell population throughout differentiation steps. These lines of evidence suggest that two functional carbohydrates, HNK-1 and Lewis x, are alternatively expressed, and that the biosynthesis of the HNK-1 carbohydrate epitope may be regulated by the expression of the fucose residue on GlcNAc. The X-ray crystal structure of GlcAT-P, and the modeled structure of a complex of GlcAT-P and Lewis x also revealed that it was almost stereochemically impossible to gain access to the enzyme when a fucose residue was on GlcNAc (Kakuda et al., 2004b
).
We examined the acceptor substrate specificity toward oligosaccharides. GlcAT-P exhibits high specificity toward type 2 (Galß1-4GlcNAc) glycans. This finding was in good agreement with in the case of the native GlcAT-P purified from rat brain (Oka et al., 2000; Terayama et al., 1998
). On the other hand, GlcAT-S transferred GlcA not only to type 2 but also to type 1 (Galß1-3GlcNAc) glycans and showed the highest activity toward triantennary N-linked oligosaccharides, indicating that GlcAT-S seems to have a wide specificity range. This may indicate that GlcAT-P and GlcAT-S are involved in the biosynthesis of structurally and functionally different HNK-1 carbohydrates.
In situ hybridization on the rat embryo revealed that GlcAT-S transcripts were expressed in the pallidum and retina, in which GlcAT-P expression was not specifically observed (Shimoda et al., 1999). In rat postnatal day 8 cerebellum, GlcAT-S mRNA was expressed in the internal and external granule layers. On the other hand, the expression of GlcAT-P was mainly localized in the Purkinje cell layer (unpublished data). Furthermore, although the HNK-1 carbohydrate disappeared almost completely in GlcAT-P-deficient mice, a trace of HNK-1 immunoreactivity remained on the surfaces of the soma and proximal dendrites of a subset of neurons in the perineuronal nets (Yamamoto et al., 2002
). These lines of evidence suggested that although these two GlcAT activities overlap in part, they are not functionally redundant in the brain. It is possible that there are structurally and functionally different HNK-1 carbohydrate epitopes in vivo. From this point of view, GlcAT-P-deficient mice constitute a useful tool for clarifying the structural and functional roles of the remaining HNK-1 carbohydrate epitope.
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Materials and methods |
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Preparation of expression constructs of soluble forms of GlcAT-P and GlcAT-S
For the construction of expression vectors to produce soluble forms of GlcAT-P and GlcAT-S fused with protein A, pGIR201protein A was digested with NheI. The NheI DNA fragment encoding the insulin signal sequence and IgG-binding domain of protein A was subcloned into a mammalian expression vector, pEF-BOS, which had been digested with XbaI. The resulting vector, named pEF-protA-BOS, was used for subcloning of the following truncated forms of GlcAT-P and GlcAT-S. A truncated form of GlcAT-P lacking the amino-terminal 35 amino acids was amplified by polymerase chain reaction (PCR) with rat GlcAT-P cDNA (Terayama et al., 1997) in pEF-BOS as a template using primers (5'-CTCGGATCCGTCTGGCACCAGAGCA-3' and 5'-ATTGGATCCTGTGTAGTTTCAGATCTCCACCGA-3') containing BamHI sites (underscored). A truncated form of GlcAT-S, lacking the amino-terminal 23 amino acids, was amplified by PCR with rat GlcAT-S cDNA (Seiki et al., 1999
) in pEF-BOS as a template using primers (5'-ATCAGATCTGACGTGGACCCCCGCA-3' and 5'-CGTAGATCTGCGGAGAAGCGGAACGA-3') containing BglII sites (underscored). Each PCR product was digested with BamHI or BglII and then inserted into pEF-protA-BOS. The recombinant plasmids with the correct orientation, pEF-protA-GlcAT-P and pEF-protA-GlcAT-S, were used for the following experiments.
Preparation of soluble forms of GlcAT-P and GlcAT-S
COS-1 cells (5 x 106 cells) were transfected with pEF-protA-GlcAT-P or pEF-protA-GlcAT-S (10 µg each) using lipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 24 h incubation, the culture medium was replaced with serum-free ASF104 medium (Ajinomoto, Tokyo, Japan), followed by incubation for another 4 days. The enzymes secreted into the medium were adsorbed to normal rabbit IgG Sepharose 4B (Amersham Bioscience, Little Chalfont, U.K.), which was then used as the enzyme sources.
Western blot analysis of soluble forms of GlcAT-P and GlcAT-S
The protA-GlcAT-P and protA-GlcAT-S purified with IgG Sepharose 4B were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) (520%) and then transferred to nitrocellulose membranes. The blots were then incubated with HRP-conjugated rabbit IgG (Zymet, San Francisco, CA) and visualized with a DAB substrate kit (Funakoshi, Tokyo, Japan).
GlcAT assay for glycoprotein acceptors
The GlcAT activity toward glycoprotein acceptors was measured essentially as described previously (Oka et al., 1992) with slight modification. An equivalent amount of each enzyme was incubated at 37°C for 3 h in a reaction mixture with a final volume of 50 µl comprising 200 mM MES, pH 6.5, 0. 2% NP-40, 20 mM MnCl2, 20 µg ASOR, 100 µM UDP-[14C]-GlcA (200,000 dpm), and 0.5 mM ATP. After incubation, the assay mixture was spotted onto a 2.5-cm Whatman No.1 disc. The disc was washed with a 10% (w/v) trichloroacetic acid solution three times, followed by with ethanol/ether (2:1, v/v) and then with ether. The disc was air-dried, and then the radioactivity of [14C]-GlcA-ASOR on it was counted with a liquid scintillation counter (Beckman LS-6000).
GlcAT assay for glycolipid
GlcAT activity toward a glycolipid acceptor was also measured essentially as described previously (Kawashima et al., 1992) with slight modification. An equivalent amount of each enzyme was incubated at 37°C for 3 h in a reaction mixture with a final volume of 50 µl comprising 80 mM sodium cacodylate buffer, pH 6.0, 0.4% NP-40, 10 mM MnCl2, 7.5 µg paragloboside, 100 µM UDP-[14C]-GlcA (200,000 dpm), and 10 mM ATP. The reaction was terminated by the addition of 1 ml chloroform/methanol (2:1, v/v). The radioactive reaction products were separated from labeled precursors by passage through Sephadex G-25 superfine column (1 ml), which had been equilibrated with chloroform/methanol/water (60:30:4.5, v/v/v), and then the radioactivity of [14C]-GlcA-paragloboside, eluted at the void volume of the column, was counted with a liquid scintillation counter (Beckman LS-6000).
GlcAT assay for oligosaccharides
GlcAT activity toward oligosaccharides was measured as follows: An equivalent amount of each enzyme was incubated at 37°C for 3 h in a reaction mixture with a final volume of 50 µl comprising different concentrations of oligosaccharides, 200 mM MES (pH 6.5), 0.2% NP-40, 20 mM MnCl2, 100 µM UDP-[14C]-GlcA (200,000 dpm), 0.5 mM ATP, and 2 µl heat-inactivated NonidetP-40 extract of rat brain (13). After incubation, the reaction was terminated by the addition of 1 ml 5 mM phosphate buffer, pH 6.8. The radioactive reaction products were separated from UDP-[14C]-GlcA by passage through anion exchange resin AG1-X4 column (1 ml) that had been equilibrated with 5 mM phosphate buffer, pH 6.8. The column was washed with 5 ml of 5 mM phosphate buffer, pH 6.8, and then the effluent and washings were collected. The radioactivity was counted with a liquid scintillation counter (Beckman LS-6000).
Preparation of soluble NCAM as a glycoprotein substrate
The expression construct of Fc-tagged rat NCAM (Kakuda et al., 2004a) was transfected into Lec2 cells using lipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 24 h incubation, the culture medium was replaced with ASF104 medium (Ajinomoto), followed by incubation for another 5 days. The NCAM-Fc secreted into the medium was collected with a protein G Sepharose column. The adsorbed NCAM-Fc was eluted with 0.1 M citrate buffer, pH 2.2; neutralized immediately with 1 M TrisHCl buffer, pH 9.0; and then concentrated before use as a glycoprotein substrate. The purify of the NCAM-Fc was checked by western blot analysis after SDSPAGE under reducing conditions. The membrane was incubated with 5 µg/ml of biotin-conjugated ricinus communis agglutinin or rabbit anti-human Fc antibodies. After incubation with HRP-conjugated avidin or anti-rabbit IgG, the membrane was visualized with a DAB substrate kit (Funakoshi).
Amino acid analysis
NCAM-Fc and ASOR were hydrolyzed in 6 N HCl at 100°C for 16 h, and then the amino acid and amino sugar contents were determined with an amino analyzer (Hitachi L8500).
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Acknowledgements |
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Abbreviations |
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References |
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