Glycobiology Advance Access originally published online on February 5, 2008
Glycobiology 2008 18(4):290-302; doi:10.1093/glycob/cwn007
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A novel 
1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures
2 Department of Biochemistry and Molecular Biology;
3 The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA;
4 Department of Molecular Cell Biology and Immunology, Glycoimmunology Group, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
1 To whom correspondence should be addressed: Tel: +1-404-727-5962; Fax: +1-404-727-2738; e-mail: rdcummi{at}emory.edu
Received on May 14, 2007; revised on January 23, 2008; accepted on January 29, 2008
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Abstract |
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The
1,2-fucosyltransferase family (1,2FT) is the largest family of glycosyltransferases in the genome of the free-living nematode Caenorhabditis elegans, and early evidence suggests that each member may have a unique activity. Here we describe a C. elegans gene (designated CE2FT-2) encoding an 1,2FT that has the potential to generate the sequence Fuc1-2Galβ1-3GalNAc-R, which is the H-type 3 blood group structure. The CE2FT-2 cDNA encodes a putative transmembrane protein that shows 
42% amino acid identity to a previously cloned C. elegans 1,2FT (termed CE2FT-1), but has a very low identity (16–20%) to 1,2FT sequences in humans, rabbits, and mice. A recombinant form of CE2FT-2 expressed in human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP, but unexpectedly, the enzyme is inactive toward the acceptor Galβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTs previously described from animals that all utilize Galβ-O-phenyl. CE2FT-2 is expressed at all stages of worm development, but remarkably, promoter analysis of the CE2FT-2 gene using green fluorescent protein reporter constructs indicates that the CE2FT-2 is expressed exclusively in pharyngeal cells of the worm from embryo to an adult stage. Because pharyngeal cells are known to secrete their glycoconjugates to the nematode surface, these results may indicate that products of CE2FT-2 contribute to interactions of the nematode with its environment or are used as ligands for bacterial attachment. These findings, along with those on other 1,2FTs in C. elegans, suggest that each 1,2FT in this organism may have a unique acceptor specificity, expression pattern, and biological function. Key words: Caenorhabditis elegans / fucosylation / fucosyltransferase / gene / green fluorescent protein
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Fucose residues in N- and O-glycans of glycoproteins and in glycosphingolipids are important in many aspects of glycan function and recognition by carbohydrate-binding proteins and pathogens. In animals fucose linkages to other sugar residues include Fuc
1-2-R, Fuc1-3-R, Fuc1-4-R, and Fuc1-6-R on both extracellular (Lowe 1993
, 1997; Oriol et al. 1999) and some cytosolic glycoproteins (van Der Wel et al. 2001). But within each linkage class of fucose, there are unique and specific fucosylated oligosaccharides that are structurally and functionally different. For example, fucose is present in the human H-blood group antigen (the H-disaccharide Fuc1-2Galβ1-R) and occurs in four potentially different structures that are immunologically distinct, known as H-type 1 (Fuc1-2Galβ1-3GlcNAc-R), H-type 2 (Fuc1-2Galβ1-4GlcNAc-R), H-type 3 (Fuc1-2Galβ1-3GalNAc1-R), and H-type 4 (Fuc1-2Galβ1-3GalNAcβ1-R) antigens (Clausen and Hakomori 1989). While the functions of such H-antigens in humans are still puzzling, it is interesting that H-antigens are recognized by various pathogens, including viruses, e.g. those within the Norovirus group (Harrington et al. 2002; Marionneau et al. 2002; Hutson et al. 2002, 2003; Huang et al. 2003), and bacteria, including Campylobacter jejuni (Ruiz-Palacios et al. 2003) and Helicobacter pylori (Alkout et al. 1997). In addition, H-antigen expression in intestinal cells is upregulated by microbial infection (Bry et al. 1996; Lin et al. 2001; Ruiz-Palacios et al. 2003). Infectious organisms, such as H. pylori (Wang et al. 1999), also synthesize H-antigen where it is involved in host interactions.
The synthesis of fucosylated glycans results from expression of specific fucosyltransferase genes. Many species contain a large number of genes encoding fucosyltransferases (Javaud et al. 2003). In particular, a startling result of the complete sequencing of the genome of Caenorhabditis elegans, a free-living nematode, is the discovery of nearly two dozen genes encoding putative fucosyltransferases, most of which are predicted to be 1,2-fucosyltransferases (1,2FTs) (Oriol et al. 1999) and 1,3-fucosyltransferases (Paschinger et al. 2004, 2005; Nguyen et al. 2007) (also see Family GT11 at the website of the Consortium for Functional Glycomics accessed at http://www.functionalglycomics.org). C. elegans offers an attractive model system in which to explore the potential roles of fucosylated glycans in animal development.
We reported earlier that a major 1,2FT activity in extracts of adult C. elegans generates the H-type 3 antigen Fuc1-2Galβ1-3GalNAc1-R (Zheng et al. 2002). Here we report the identification and characterization of this unique 1,2FT (CE2FT-2) from C. elegans capable of synthesizing the H-type 3 glycan antigen from the acceptor Galβ1-3GalNAc1-R and related structures and an analysis of its unique cellular expression during C. elegans development.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Identification and isolation of a C. elegans cDNA encoding a novel
1,2-fucosyltransferaseWe previously used a panel of acceptors to assay total extracts of C. elegans for possible fucosyltransferase activities with radioactive GDP-Fuc as the donor. The highest activity was found toward the vertebrate O-glycan-related structure Galβ1-3GalNAc
-O-pNP and we confirmed that in C. elegans extracts the enzyme capable of fucosylating this acceptor is an 1,2FT (Zheng et al. 2002). To identify the cognate C. elegans gene encoding 1,2FT activity toward Galβ1-3GalNAc-O-pNP, we screened a number of C. elegans genes encoding putative 1,2FTs (see Family GT11 at the website of the Consortium for Functional Glycomics at http://www.functionalglycomics.org). One of the genes identified was harbored in C. elegans cosmid F08A8 (GenBank accession no. Z99710), derived from chromosome I. This gene predicts a cDNA of 1152 bp encoding the hypothetical protein F08A8.5, which shows 42% amino acid identity to the 1,2-FT CE2FT-1(Zheng et al. 2002). To isolate this putative 1,2FT cDNA, gene-specific primers were designed to amplify the entire coding sequence of the C. elegans F08A8.5 from total synthesized C. elegans cDNA, as described in the Materials and methods section. Following amplification, the obtained 1.2-kb PCR product was cloned into the pCR3.1 vector. Three clones containing inserts were selected and nucleotide sequencing confirmed that they contained the full-length in-frame cDNA (Figure 1A) that correctly matched the predicted cDNA from the database. However, we observed the presence of an extra 57-bp in-frame sequence, beginning at nucleotide 154 and ending at nucleotide 210 (Figure 2A) that had previously been predicted by the database to be an intron. We subsequently isolated six other clones and they all contained this extra 57-bp in-frame sequence.
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The identified putative C. elegans
1,2FT gene spans 12 exons (Figure 1B) and its corresponding cDNA contains a full-length open-reading frame that encodes a putative protein with 402 amino acids, designated as CE2FT-2 (Figure 1A). There are four predicted N-glycosylation sites at Asn residues 43, 119, 175, and 301 within the N-glycosylation sequon Asn-X-Ser/Thr where X 
Pro. The Kyte–Doolittle hydropathy analysis (Kyte and Doolittle 1982) of the protein sequence suggests the presence of a 23-amino-acid transmembrane domain at the N-terminus (Figure 1C). Thus, the encoded protein displays the typical hallmarks of most Golgi-localized glycosyltransferases and would be predicted to be a type-II membrane protein with a cytosolic N-terminus and a Golgi lumenal C-terminus. The cloned cDNA for CE2FT-2 shows 42% sequence identity to the previously cloned C. elegans 1,2FT, CE2FT-1 (Zheng et al. 2002), but has a very low identity (16–20%) at the amino acid level to the 1,2FT sequence in humans, bovine, and mice (Figure 2B). All so-called "-1,2-motifs" (motifs I, II, and III) identified previously in CE2FT-1 (Zheng et al. 2002) were conserved at similar locations in the C-terminal region of CE2FT-2, although motif II was less well preserved than motifs I and II.
Expression of C. elegans CE2FT-2 cDNA in 293T cells identifies the recombinant protein as an 1,2FT able to fucosylate Galβ1,3GalNAc-R
To define whether the cloned C. elegans cDNA encodes a functional 1,2FT, we made a cDNA construct encoding CE2FT-2 full-length protein with and without a C-terminal HPC4-epitope tag (Stearns et al. 1988; Rezaie et al. 1992). This construct was inserted into the mammalian expression vector pcDNA4/HisMaxC and the resulting plasmid (pcDNA4/ HisMaxC/CE2FT-2T) was transiently transfected into human 293T cells. The expression of the recombinant protein was confirmed by SDS–PAGE and Western blotting, using an antibody directed to the HPC4 epitope. The immunoblot (Figure 3 – inset) revealed a protein band with apparent MW of 53–61 kDa, which would be expected of a variably glycosylated polypeptide of 52.6 kDa (predicted from the full-length polypeptide with the 12-amino-acid HPC4-epitope tag). The presence of possible N-glycans on the enzyme was examined in stable cell constructs, as described below.
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Extracts of mock-transfected cells showed background levels of a fucosyltransferase activity toward the acceptor Galβ1-3GalNAc
-O-p-nitrophenol (pNP), but cells expressing the HPC4-tagged full-length CE2FT-2T showed an 8-fold increased activity toward this acceptor (Figure 3). Moreover, the fucosyltransferase activity was highly enriched on beads containing an immobilized HPC4 monoclonal antibody, whereas no activity was observed using HPC4-beads incubated with mock-transfected cells as the enzyme source (Figure 3). These results demonstrate that CE2FT-2T fucosylates the acceptor Galβ1-3GalNAc-O-pNP.
To define the linkage of fucose in the reaction product, the radiolabeled oligosaccharides generated by recombinant CE2FT-2 and the acceptor Galβ1-3GalNAc-O-pNP using GDP-[3H]fucose as the donor was isolated and treated with either 1,2-, 1,3/4-, or 1,6-fucosidase. The treated material was reisolated by adsorption to Sep-Pak C-18 cartridges. The results shown in Figure 4 indicate that 3H-fucose was released from the fucosylated product only by 1,2-fucosidase, but no release occurred with either 1,3/4-fucosidase or 1,6-fucosidase. These results demonstrate that the fucosylated reaction product obtained with CE2FT-2 has the sequence Fuc1-2Galβ1-3GalNAc-O-pNP. In addition, these data identify the cloned fucosyltransferase as a GDP-Fuc:Galβ1-3GalNAc-R 1,2-fucosyltransferase.
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Acceptor specificity of recombinant CE2FT-2
To further characterize the specificity of CE2FT-2, we incubated different acceptors with the donor GDP-[3H]fucose and the recombinant form of CE2FT-2 expressed in human 293T cells. The highest FT activity (Table I) was found toward Galβ1-3GalNAc
-O-pNP and the related structures Galβ1-3(GlcNAcβ1-6)GalNAc-O-pNP and Galβ1-3GalNAc. The low activity was observed toward Galβ1-3GlcNAcβ-O-pNP and Galβ1-3(Fuc1-4)GlcNAc. All known animal 1,2FTs reported to date are active toward Galβ-O-pNP and most simple acceptors with the terminal β-linked Gal residue (Kumazaki and Yoshida 1984; Ernst et al. 1989; Rajan et al. 1989; Sarnesto et al. 1992; Kelly et al. 1995; Lin et al. 2001). Unexpectedly, no significant activity was observed toward either Galβ-O-pNP or Gal-O-pNP (Table I). Thus, CE2FT-2 shows unique acceptor specificity compared to previously characterized 1,2FTs. In addition, no activity was detected toward any acceptor tested with a terminal β1-4- or β1-6-linked Gal. For example, the enzyme is unable to transfer fucose to Galβ1-4Xylβ-O-benzyl, which was an excellent acceptor for CE2FT-1 (Zheng et al. 2002). Also, CE2FT-2 does not appear to transfer fucose to another fucose residue, as occurs within the structure Galβ1-3(Fuc1-4)GlcNAc to form the unusual Fuc1-2Fuc1-R, a difucosyl sequence known to occur in the parasitic helminth trematode Schistosoma mansoni (Khoo et al. 1995; 1997). This tentative conclusion is consistent with our observation that the presence of fucose reduced the acceptor activity of Galβ1,3(Fuc1,4)GlcNAc compared to Galβ1,3GlcNAc, thus making it unlikely that CE2FT-2 transfers Fuc to another Fuc residue. CE2FT-2 showed a relatively good activity toward Galβ1-3(GlcNAcβ1-6)GalNAc-O-pNP and Galβ1-3GalNAc, but acted poorly toward Galβ1-3GlcNAcβ-O-pNP. Thus, we conclude that this enzyme recognizes acceptors with terminal Gal and penultimate GalNAc. The possibility that the enzyme may recognize glycopeptides carrying the O-linked core 1 structure was investigated using a synthetic glycopeptide 4-GP-2 which contains the Galβ1-3GalNAc1-O-Thr sequence [Glu-Tyr-Glu-Tyr-Leu-Asp-Tyr-Asp-Phe-Leu-Pro- Glu-(Galβ1-3GalNAc1-O-Thr)-Glu-Pro-Pro-Glu-Met]. Surprisingly, CE2FT-2 is inactive toward 4-GP-2. This may be due to the close proximity of the peptide sequence. Thus, CE2FT-2 may act on acceptors Galβ1-3GalNAc-R, in which R represents a longer oligosaccharide structure allowing access of the enzyme to the terminal sequence, and/or may include a hydrophobic part.
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The dependence of purified, recombinant CE2FT-2 captured on HPC4-beads on acceptor concentration for Galβ1-3GalNAc
-O-pNP was determined. The enzyme exhibited an acceptor Km of 0.61 mM in the presence of 50 µM GDP-Fuc (Figure 5A). The dependence on GDP-Fuc was also determined and the apparent Km was estimated to be 1 mM (Figure 5B). We estimated stability of the product and sugar nucleotide donor in these reactions and found that they were stable without significant degradation, so the relatively high Km values are not a reflection of product or donor loss and suggest that CE2FT-2 has relatively high Km toward both the acceptor and donor.
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Recombinant CE2FT-2 carries N-glycans
The amino acid sequence of CE2FT-2 predicts 4 potential N-glycosylation sites (Figure 1B). To assess whether the 293T-derived CE2FT-2 contains N-glycans, we prepared 293T cells that stably expressed the HPC4-tagged recombinant CE2FT-2 (CE2FT-2T). The recombinant CE2FT-2T was purified from the cells and treated with PNGase F and Endo-H respectively to remove potential N-glycans. PNGase F cleaves the R-GlcNAcβ-
Asn-R’ linkage of glycans to Asn, whereas endo-H cleaves between GlcNAc residues of R-GlcNAcβ4-GlcNAc-Asn-R in the core of hybrid- and high mannose-type N-glycans. The treated proteins were analyzed by reducing SDS–PAGE (4–20%) and Western blotting using a HPC4 monoclonal antibody. The results show that purified recombinant CE2FT-2 has an apparent Mr of 55/57 kDa (doublet) (Figure 6A and B). The glycoprotein is sensitive to both PNGase F and endo-H and the apparent Mr of the resulting proteins closely matched that predicted from the primary protein sequence (53 kDa) (Figure 6A). These results indicate that recombinant CE2FT2 from human 293T cells contains primarily hybrid- and/or high mannose-type N-glycans. The shift in apparent Mr from 55/57 kDa to 53 kDa suggests the presence of more than one N-glycan, but the number of N-glycans and the specific site of modification of the protein were not examined further. Many vertebrate glycosyltransferases appear to occur as disulfide bonded dimers or oligomers (Taniguchi et al. 1982; Eppenberger-Castori et al. 1989; Zhu et al. 1997; Qian et al. 2001; Ju, Brewer, et al. 2002; Ju, Cummings, et al. 2002). Nonreducing and reducing SDS–PAGE analyses of purified full-length HPC4-tagged CE2FT-2 indicated that CE2FT-2 has electrophoretic behavior consistent with its occurrence as a monomer, dimer, trimer, and tetramer under nonreducing conditions (Figure 6B).
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Expression of CE2FT-2 mRNA at various stages in C. elegans development
To examine the pattern of expression of CE2FT-2 and whether it is expressed during development, we analyzed the mRNA expression level at the different stages by quantitative real-time (RT)-PCR using equivalent amounts of total RNA from different developmental stages including L1, mixed L2–4 stage larvae, and young adult worms. The CE2FT-2 gene is expressed at all developmental stages with little relative difference in expression compared to
-actin (Figure 7A). Agarose gel electrophoresis indicated a single product of the expected size of 150 bp at each stage analyzed (Figure 7B).
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Expression of a CE2FT-2 promoter at various stages in C. elegans development
To localize the expression of the CE2FT-2 gene, we used reporter promoter constructs driving expression of the coelenterate green fluorescent protein (GFP) in the vector pPD95.67/CE2FT-2-prom (see the Materials and methods section), which we termed the CE2FT-2::GFP reporter. Expression of the GFP reporter gene in the transgenic worms injected with the CE2FT-2::GFP reporter was examined (Figure 8A–D). The results indicate that the promoter for CE2FT-2 is specifically activated in pharyngeal cells and is expressed at the embryonic stage (Figure 8E, F). Promoter function was evident through all developmental stages with a highly restricted GFP expression in the pharyngeal neurons and gland cells (Figure 8A–F) based on visual observations.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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We describe the identification and molecular cloning of a C. elegans cDNA encoding a new type of
1,2-fucosyltransferase and designate the corresponding C. elegans gene CE2FT-2. This gene contains an open-reading frame encoding a predicted protein of 402 amino acids with a domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression of the corresponding cDNA in human 293T cells resulted in an enzyme with the high 1,2FT activity toward the Galβ1-3GalNAc-O-pNP acceptor with GDP-Fucose as the donor. A comprehensive analysis of the specificity of CE2FT-2 shows that this enzyme has a highly restricted acceptor usage that is completely different from all known 1,2FTs to date, such as the human H-type and secretor 1,2FTs and C. elegans CE2FT-1. Remarkably, and in contrast to all known 1,2FTs, the recombinant CE2FT-2 is inactive toward the simple monosaccharide acceptor Galβ-O-pNP. Our data suggest that CE2FT-2 requires Galβ1,3GalNAc as a minimal acceptor structure. Further extension of this minimal structure, such as modification of the GalNAc by a β1,6GlcNAc, or by adding a hydrophobic group to its reducing end, is allowed, whereas other modifications such as -linkage of the Galβ1,3GalNAc to a peptide, thus forming the common mammalian type 1 O-glycan core structure known as T-antigen blocks an enzyme activity. The observation that the enzyme acts more efficient on Galβ1-3GalNAc-O-pNP as an acceptor than that on the free oligosaccharide Galβ1-3GalNAc indicates that the enzyme may have a preference for a hydrophobic group at the reducing end of acceptors or may prefer the -configuration, since the free disaccharide is in equilibrium between reducing and β configurations in solution. Thus, in vivo the enzyme may prefer acceptors with a hydrophobic character, such as glycolipids or glycopeptides with a hydrophobic peptide region. Alternatively, the enzyme may have a preference for terminal Galβ1-3GalNAc sequences that are -linked to another sugar, thus generating H-type 3 rather than H-type 4 structures as a part of larger glycan molecules.
Previously we showed that the 1,2FT enzyme activity toward Galβ1-3GalNAc-R represents a major 1,2FT activity in C. elegans. This is remarkable since our data here indicate that the expression pattern of CE2FT-2 is restricted to pharyngeal cells. It may be that these cells synthesize oligosaccharides with terminal type 3 or type 4 H-antigens in significant amounts, but it cannot be excluded that one or more of the other putative C. elegans 1,2FTs encodes an enzyme with identical or similar specificity in other cell types. The restrictive expression patterns and enzyme activities of CE2FT-1 and CE2FT-2 suggest that each of the 22 putative 1,2-fucosyltransferases in C. elegans may have unique roles in development and/or homeostasis of the nematode. The lack of terminal sialylation in C. elegans may be compensated by more extensive and diverse types of fucosylation.
In mammals this acceptor Galβ1-3GalNAc-R for CE2FT-2 is found as a part of the core of gangliosides, which is further modified by one or more sialic acid residues. In addition, the Galβ1,3GalNAc-R structure represents a common O-linked core structure, the T-antigen (core 1), which is usually extended, or directly sialylated, in mammals. In C. elegans, however, no sialic acids have been demonstrated thus far, and it may be possible that such core structures are instead fucosylated. Indeed, type 3 or type 4 H-antigens have been found in invertebrate glycoproteins. For example, the Fuc1,2Galβ1,3GalNAc moiety is found as an internal motif in glycolipids of C. elegans (Griffitts et al. 2005), as well as on N-glycans of Lymnaea stagnalis haemocyanin (Van Kuik et al. 1987). Interestingly, O-glycans containing variously methylated forms of the Fuc1,2Galβ1,3GalNAc moiety have been demonstrated in organisms strongly related to C. elegans, such as species of the pathogenic nematode Toxocara. In Toxocara canis these moieties can be mono- or di-O-methylated and these represent the major O-glycan types of excretory–secretory antigens (Khoo et al. 1991). Mice infected with T. canis generate antibodies not only toward the methylated glycan antigens, but also to the unmethylated H-type 3 glycan epitope, indicating that both structures are present in T. canis (Schabussova et al. 2007). Although in our assays a simple O-glycopeptide could not act as an acceptor for CE2FT-2, we cannot rule out the possibility that additional factors, such as a hydrophobic peptide sequence or extended O-glycans, may be required to yield a good O-glycan acceptor.
Quantitative RT-PCR analysis indicated that the CE2FT-2 is expressed throughout worm development in all stages with a little difference. Promoter analysis of the CE2FT-2 gene using green fluorescent protein reporter constructs indicated that the CE2FT-2 promoter is expressed exclusively in pharyngeal neural and gland cells of the worm, from embryo to adulthood. Because pharyngeal gland cells can secrete their glycoconjugates to the cuticle and surrounding surfaces (Kramer 1997), these expression results may indicate a role for CE2FT-2 in 1,2-fucosylation of glycans at the nematode surface, where they may contribute to interactions of the nematode with its environment. Interestingly, a C. elegans mutant (srf-3) has been described recently that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis. SRF-3 was characterized as a UDP-galactose transporter of the Golgi apparatus occurring in pharyngeal cells, and the mutant showed a reduced expression of both fucosylated N-, and to a less extent, O-glycans (Cipollo et al. 2004; Hoflich et al. 2004). Based on the expression of both SRF-3 and CE2FT-2 in pharyngeal cells, this may indicate that this gland secretes glycoconjugates with Fuc1-2Galβ1-3GalNAc-R termini. Several bacteria are known to adhere to terminal 1-2-fucose, including H. pylori and P. aeruginosa. It would be interesting to investigate whether M. nematophilum and Y. pseudotuberculosis biofilm bind to glycoconjugates containing the Fuc1-2Galβ1-3GalNAc-R structure. Searching the database of the C. elegans Knockout Consortium for mutations in CE2FT-2 resulted in one existing mutant strain (VC1055), with 725 bp deletion located at the physical position 12956800–12957524, chromosome I (Genotype F08A8.5_GK453). However, this mutated strain GK453 has no apparent phenotype. In addition, multiple RNAi experiments reported in the WormBase (http://wormbase.org/db/seq/rnai?name=F08A8.5;view=all), which target a different genomic region of F08A8.5 (CE2FT-2), did not generate a demonstrable phenotype in three independent experiments. The possible loss of function of CEFT-2 could cause subtle changes, and future experiments to explore this may require extensive phenotypic analysis for cell-associated functions involving pharyngeal neural and gland cells.
The expression of CE2FT-2 in anterior pharyngeal neural cells is remarkable, especially since this expression pattern seems to be conserved in mammals. It has been shown that in rabbits the H-blood group type 1,2-fucosyltransferase (RFT-I), gene, and its biosynthetic products, H-antigens (Fuc1-2Galβ), are abundantly expressed in a subset of dorsal root ganglia (DRG) neurons (Hitoshi et al. 1998, 1999). These antigens have also been demonstrated in primary sensory neurons and their axons in human and other primates (Mollicone et al. 1986; Mori 1987; Kusunoki et al. 1991). Most of the H-positive axons of primary sensory neurons are thought to be C-fibers that mediate nociceptive or thermoceptive inputs or both. C. elegans may provide a unique model system to study the functional role of 1,2-fucosylation in neural cells.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Materials
Sodium cacodylate, MnCl2, ATP, L-fucose, Galβ-O-phenyl, GlcNAcβ-O-p-nitrophenol (pNP), Galβ1-6Galβ-O-pNP, and GalNAcβ-O-pNP were purchased from Sigma Chemical Co. (St. Louis, MO).
1,2-Fucosidase and 1,3-fucosidase were purchased from Prozyme, Inc. (San Leandro, CA). 1,6-Fucosidase and GDP-fucose were purchased from Calbiochem Co. (La Jolla, CA). Galβ-O-pNP, Gal-O-pNP, and Glcβ-Mu (methylumbelliferon) were obtained from Koch-Light Laboratories (now NBS Biologicals Ltd., Huntingdon,England). Galβ1-4Glcβ-O-pNP, Galβ1-4GlcNAc-O-Bz, Galβ1-4GlcNAcβ1-2Man-O-pNP, Galβ1-3GlcNAcβ-O-pNP, Galβ1- 3(GlcNAcβ1-6)GalNAc-O-pNP, and Galβ1-3GalNAc-O-pNP were purchased from Toronto Research Chemicals Inc. (Downsview, ON, Canada). Galβ1-3GlcNAcβ1-3Galβ1-4Glc was from Oxford GlycoSystems. Galβ1-3Galβ1-4Glc was from Dr. M. Messer (University of Sydney, NSW, Australia). Galβ1-3Galβ1-4Xyl-O-Bn and Galβ1-4Xyl-O-Bn were from Dr. T. Norberg (Swedish University of Agricultural Sciences, Uppsala, Sweden). The synthetic glycopeptide 4-GP-2 with the sequence Glu-Tyr-Glu-Tyr-Leu-Asp-Tyr-Asp-Phe-Leu-Pro- Glu-(Galβ1-3GalNAc1-O-Thr)-Glu-Pro-Pro-Glu-Met and having the GalNAc-substituted Thr, as indicated, was kindly provided by Dr. Anne Leppänen (University of Helsinki, Helsinki, Finland). GDP-[3H]Fuc or GDP-[14C]Fuc (New England Nuclear) was diluted with unlabeled GDP-Fuc to give the desired specific activity. Taq DNA polymerase and other PCR reagents were obtained form Boehringer Mannheim (Indianapolis, IN). Oligonucleotide primers were synthesized by GIBCO BRL Life technologies. The Eukaryotic TA Cloning Kit (bidirectional) and pcDNA4/HisMax vectors were obtained from Invitrogen (San Diego, CA). Restriction enzymes were purchased from New England Biolabs, Inc. (Beverly, MA).
C. elegans culture and preparation of C. elegans total RNA
Standard molecular biology procedures were used unless otherwise stated. The standard laboratory wild-type strain N2 worms were grown on NGM plates seeded with OP50 bacteria. The mixed-stage worms were washed with a M9 buffer and were frozen in liquid nitrogen for 2 min and the worm pellets were stored at –80°C. The frozen worms were ground and suspended in Ambion's Denaturation Solution (Total RNA Isolation Kit, Ambion Inc., Austin, TX). The total RNA was extracted by following the manufacturer's instruction.
Cloning and expression of CE2FT-2
The CE2FT-2 cDNA was cloned by RT-PCR. Random primed first strand cDNA was synthesized from 5 µg of total C. elegans RNA using the SuperScript Preamplification System for the First Strand cDNA Synthesis Kit (GiBcoBRL, Gaithersburg, MD). The oligonucleotides 5'-GAAACTATGA-GGTATAATTC-3' and 5'-AATCA-ATATTTGCTGATCTTTC-3' were used as forward and reverse primers, respectively, to amplify the CE2FT-2 coding sequence from the random primed first strand cDNA. Reaction mixtures for amplification by Taq DNA polymerase contained 1 µM of each primer, 200 µM dNTPs, 2.5 mM MgCl2, and 2 µL of C. elegans cDNA in a final volume of 50 µL. Amplification was carried out by an initial denaturing step of 94°C for 5 min, followed by 35 cycles of 94°C/1 min, 59°C/1 min, and 72°C/2 min. These cycles were then followed by an extension period of 72°C/7 min. Following amplification, the PCR products were separated by agarose gel electrophoresis and a single 1200-bp DNA band was excised from the gel and purified by the QIAquick Gel Extraction Kit (QIAGEN, Inc. San Clarita, CA). The purified DNA was ligated into the pCR3.1 vector (Bidirectional Eukaryotic TA Cloning Kit) and subsequently transformed into One Shot Top10F’ competent cells according to the procedures provided with the kit. Clones containing inserts in the proper orientation were selected, and nucleotide sequencing confirmed the identity of cloned inserts. A plasmid containing the expected nucleotide sequence was selected and a Kpn1/Apa1 restriction fragment containing the cDNA insert was subcloned into the vector pcDNA4/HisMax C in the proper-reading frame. The resulting plasmid (pcDNA4/HisMaxC/CE2FT-2) contained the full-length cDNA of CE2FT-2 as was confirmed by nucleotide sequencing [GenBank Accession EF015633]. Plasmid for transfection was prepared using the QIAGEN Endotoxin-Free Plasmid DNA Purification Kit.
Construction and stable expression of C-terminal HPC4-epitope-tagged CE2FT-2
A mammalian expression vector encoding CE2FT-2 with a C-terminal tag containing the 12-amino-acid Ca2+-dependent HPC4 epitope (EDQVDPRLIDGK) (Stearns et al. 1988; Rezaie et al. 1992) was constructed by PCR using pcDNA4/HisMaxC/CE2FT-2 as a template. The forward primer used was T7 primer 5'-TAATACGACTCA CTATAGGG-3'. The reverse primer (5'-CGGAATTCACTTG CCGTCGATCAGCCTGGGGTCCACCT-GGTCCTCATATT- TGC-TGATCTTTCCGTTTAGATC-3') contained the sequence encoding the HPC4 epitope immediately following the C-terminal tyrosine of CE2FT-2. The PCR was performed by denaturation at 94°C for 2 min, amplification for 25 cycles at 94°C, 1 min; 59°C, 1 min; and 72°C, 2 min. The expected 1560-bp PCR product was purified from a 1.2% TAE agarose gel, digested by Kpn I and EcoR I, and cloned in frame into pcDNA4/HisMaxC vector. The resulting plasmid (pcDNA4/His-MaxC/CE2FT-2T) contained the expected C-tagged CE2FT-2 sequence as was confirmed by nucleotide sequencing. Human 293T cells were transfected with pcDNA4/HisMaxC/CE2FT-2T plasmid using FuGene 6 Transfection Reagent, as described by the manufacturer (Boehringer Mannheim). A 293T cell line stably expressing CE2FT-2T was obtained by culturing transfected 293T cells with 600 µg/mL of Zeocin for 5 weeks. The stable transfected cells were harvested and solubilized in a 50 mM sodium cacodylate buffer containing 1% Triton X-100 and cell extracts were prepared as described above.
Preparation of HPC4-UltraLinkTM medium
Twenty-five milligrams of HPC4 monoclonal antibody (Stearns et al. 1988; Rezaie et al. 1992) was dissolved in 20 mL of 0.1 M MOPS and 0.6 M sodium citrate, pH 7.5, and coupled to 0.6 g of UltraLinkTM Bio-support medium (Pierce) at room temperature for 1 h, followed by blocking with 3 M ethanolamine (1 h, 20°C). The resin was then washed with 1 M NaCl and equilibrated with 25 mM Tris–HCl, pH 7.2, 150 mM NaCl, and 1 mM CaCl2.
Capture of the recombinant HPC4-tagged CE2FT-2 on HPC4-UltraLink Bio-support medium beads
To capture the recombinant HPC4-tagged CE2FT-2 on HPC4-UltraLink Bio-support medium beads, the cell extracts containing recombinant HPC4-tagged CE2FT-2 and 1 mM CaCl2 were incubated overnight with HPC4-UltraLink beads at 4°C. The beads were collected by centrifugation at 2000 x g for 5 min and washed three times with 50 mM Tris–HCl, pH 7.2, 1 M NaCl, and 1 mM CaCl2. The beads were then washed twice with 50 mM Tris–HCl, pH 7.2, 150 mM NaCl, and 1 mM CaCl2. In some cases the recombinant HPC4-epitope-tagged CE2FT-2 was captured on immobilized HPC4-agarose and was assayed for the activity directly, as described below, or it was recovered in a soluble form after elution with 5 mM EDTA.
Fucosyltransferase assay
The fucosyltransferase assays were performed in a 50 µL reaction mixture containing 100 mM sodium cacodylate (pH 7.0), 20 mM MnCl2, 5 mM ATP, 15mM fucose, 50 µM GDP-[3H]-fucose (40–50,000 cpm/nmol), 1 mM acceptor substrate (except for glycopeptide acceptors which were assayed at 0.15 mM), and 10–20 µL of cell extract or HPC4-UltraLink beads containing captured recombinant CEFT2. Reaction mixtures were incubated at 25°C for 2–5 h. The ATP was included to inhibit hydrolysis of GDP-Fuc. The reaction product was separated from an unincorporated label by chromatography on a 1-mL column of Dowex 1-X8 (Cl form) as described (Zheng et al. 2002). For substrates with a hydrophobic aglycon, the reaction mixture was applied to C18 Sep-Pak cartridges (Waters, Milford, MA) washed with 15 mL of water and eluted with 2 mL of 1-butanol. The radioactivity was determined by liquid scintillation counting in 10 mL of Scintiverse-BD. When the synthetic glycopeptide 4-GP-2 was used as an acceptor, the assays were conducted as above except that methanol, rather than 1-butanol, was used for the elution of product glycopeptides from the C18 Sep-Pak cartridges. Control assays lacking the acceptor substrate were carried out to correct for incorporation into endogenous acceptors, and assays with mock-transfected cells were conducted to correct for the endogenous fucosyltransferase activity.
Characterization of fucosyltransferase products
The radiolabeled product, generated by recombinant CE2FT-2 with the acceptor Galβ1-3GalNAc-O-pNP, was isolated by chromatography on Sep-Pak C-18 cartridges (Waters) to which it bound and was eluted with acetonitrile. The isolated product was treated with either H2O (mock) or 0.8 m units of 1-2-fucosidase, 1-3/4-fucosidase or 1,6-fucosidase in 20 µL of reaction buffer 5 (Prozyme, Inc.) at 37°C for 48 h respectively. Following treatment, the samples were applied to Sep-Pak C-18 cartridges, washed with water, eluted with 2 mL of 1-butanol, and the radioactivity in the bound material determined by scintillation counting.
Preparation of DNA constructs for promoter analysis
To examine the spatial pattern of CE2FT-2 expression during C. elegans development, we prepared fusion constructs of the upstream promoter sequences of CE2FT-2 fused to gene encoding the green fluorescent protein (GFP). A 829-bp fragment of a genomic sequence containing the putative promoter region immediately upstream of the initiation site for CE2FT-2 translation was obtained by PCR using C. elegans genomic DNA as template with primers – 5'-GTTGTTCATCTCGTTCTCATG-3' and 5'-AGTTTCAATTTTCAAACAC-3'. The PCR product was run on an agarose gel and the DNA band was excised and purified by the QIAquick Gel Extraction Kit. The purified DNA was ligated into pCR3.1 vector and subsequently transformed into Top10F’ competent cells. Desired clones were selected, and their plasmids isolated and sequenced as described above. A plasmid containing the expected insert was digested with Hind III/Pst I and the restriction fragment was cloned into a pPD95.67 vector (originally provided by Dr. Andrew Z. Fire, Carnegie Institute of Washington, Baltimore, MD) thus creating plasmid pPD95.67/CE2FT-2-prom.
Preparation of transgenic worms and promoter analysis
DNA injection into the C. elegans germ line was carried out as described (Mello et al. 1991; Mello and Fire 1995). Transgenic lines were established from F2 descendants of animals injected with 10 ng/µL of CE2FT-2::GFP reporter constructs (pPD95.67/CE2FT-2-prom) and pBX, which carries the pha-1 gene, serves as a transformation marker. The L1, L2–L4, and young adult stages of N2 worms were prepared as described previously (Zheng et al. 2002).
Quantitative RT-PCR analysis of C. elegans CE2FT-2 mRNA during development
Quantitative RT-PCR was performed using an ABI Prism 7700 sequence detection (PerkinElmer Life Sciences, Waltham, MA) using the double-stranded DNA binding dye, SYBR Green. The total RNA from staged synchronous populations and the random primed first strand cDNAs were prepared as described (Zheng et al. 2002). The first strand cDNA was used as a template in PCR reactions to amplify cDNAs encoding CE2FT-2 transcripts using the oligonucleotides 5'-AAAATAACTCTGG-GATCCAGTCATATTT-3' and 5'-TTTGG-CTGGGACTTTTGTGAA-3' in the reactions containing 3 µL of first strand cDNA in a volume of 50 µL. For controls, a C. elegans actin gene-specific primer pair 5'-ATCGTCCTCGACTCTGGAGAT-3' and 5'-TCACGTCCAGCCAAGTCAAG-3' was used to amplify C. elegans actin gene under identical conditions. Cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. To confirm the absence of nonspecific amplification, the PCR products were analyzed by 1.2% agarose gel electrophoresis.
Peptide:N-glycosidase F (PNGase F) and endoglycosidase (Endo-H) digestion of recombinant of CE2FT-2
Purified HPC4-tagged CE2FT-2 was treated with one unit of either PNGase F (New England Lab) or Endo-H (Glyco) at 37°C for 24 h. After the treatment, the reaction mixtures were separated by SDS–PAGE (4–20%) and analyzed by Western blotting using the HPC4 monoclonal antibody.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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National Institutes of Health (RO1 CH/HD54832-01 to RDC)
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We would like to thank Tongzhong Ju, Kwame Nyame, Kenneth Hatter, and Wietske Schiphorst for their help and technical support in these studies. We especially thank Robert Barstead of the Oklahoma Medical Research Foundation for his advice and help.
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Abbreviations |
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CE2FT-2, C. elegans
1,2-fucosyltransferase 2; DRG, dorsal root ganglia; FT, fucosyltransferase; GFP, green fluorescent protein; PCR, polymerase chain reaction; pNP, p-nitrophenol |
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