Glycobiology Advance Access originally published online on March 16, 2007
Glycobiology 2007 17(6):586-599; doi:10.1093/glycob/cwm023
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Molecular cloning and characterization of the Caenorhabditis elegans 
1,3-fucosyltransferase family
3 Department of Biochemistry and Molecular Biology
4 Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center
5 Program in Molecular, Cell, and Developmental Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
6 Department of Molecular Cell Biology, 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 October 3, 2006; revised on February 21, 2007; accepted on February 22, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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The genome of Caenorhabditis elegans encodes five genes with homology to known
1,3 fucosyltransferases (1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans 1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Man1-3(Man1-6)Manß1-4GlcNAcß1-4(Fuc1-3)GlcNAcß1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galß1-4(Fuc1-3)GlcNAcß1-3Galß1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 °C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core 1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the 1,3FTs in C. elegans has unique specificity and expression patterns. Key words: C. elegans / fucosyltransferase / cloning / N-glycans / neuronal cells
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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The biological functions associated with glycoproteins and oligosaccharides are often associated with terminal elements at the non-reducing end of glycans. These termini typically contain fucose and sialic acid residues, and such residues are commonly part of the glycan determinants recognized by glycan-binding proteins. Fucose, unlike sialic acid, which is absent in many invertebrates and in plants, is one of the most common terminal sugars in glycan determinants. Fucosylated glycans and fucosyltransferases (FTs) have been found in mammals, insects, helminthes, prokaryotes, and plants (Staudacher et al. 1999
). Their widespread occurrence in nature emphasizes their importance during molecular evolution. Fucosylated glycans participate in a wide variety of cellular recognition events such as recognition by selectins in the recruitment of leukocytes to sites of infection and their homing to lymph nodes (Lasky 1995; Lowe 1997; McEver and Cummings 1997; Vestweber and Blanks 1999; Lowe and Marth 2003), tumor metastasis (Hakomori et al. 1984; Kannagi et al. 1986; Nakamori et al. 1993), neural development (Ashwell and Mai 1997; Allendoerfer, et al. 1999), angiogenesis (Nguyen et al. 1993), fertilization (Johnston et al. 1998), signal transduction (Artavanis-Tsakonas et al. 1999), and bacterial adhesion (Ilver et al. 1998).
Fucosylated glycans are generated by FTs, which are a group of enzymes responsible for the transfer of fucose from guanosme 5-diphosphate–(1–3,4) fucosidase (GDP–Fuc) to oligosaccharide acceptors linked to protein and lipid. They are classified based on their linkage types, which include the 1,2-, 1,3/4-, and 1,6-FTs. The enzymes all appear to be type II transmembrane proteins with a short N-terminal cytoplasmic domain, a signal membrane anchor domain, a stem region, and a large globular C-terminal catalytic domain inside the luminal trans-Golgi compartment (Paulson and Colley 1989; Joziasse 1992). A review of FTs in 1999 by Oriol et al. (1999) identified 78 putative FT genes in all public sequence databases (NCBI GenBank), and the numbers have since doubled to over 150. Despite the growing understanding of the functions of fucosylated glycans, the biological functions of such large numbers of FT genes and their cognate glycan structures are largely unknown. While the loss of glycosyltransferase expression often results in severe phenotypic changes and embryonic death (Ioffe and Stanley 1994; Metzler et al. 1994; Lowe and Marth 2003; Xia et al. 2004), knockouts of many types of glycosyltransferases have mild, if any, phenotypes (Hennet et al. 1995; Priatel et al. 1997; Kolber-Simonds et al. 2004).
The Caenorhabditis elegans model system offers the opportunity to study these potential glycan functions because of its well-defined development and physiology, as well as its fully defined genomic sequence (Consortium, C.e.S. 1998). In addition, C. elegans is amenable to experimental manipulation, mutagenesis, and genetic analysis (Barstead and Waterston 1991; Fire et al. 1998). C. elegans shares many of the biological and biochemical pathways with higher organisms, and its genome revealed coding sequences for many known glycosyltransferases, except for sialyltransferases. A number of C. elegans glycosyltransferases have been cloned and characterized from our lab and others, such as N-acetylgalactosamine (GlcNAc) transferases (Chen et al. 1999, 2002; Warren et al. 2001, 2002), GalNAc-transferases (Hagen and Nehrke 1998; Kawar et al. 2002), FTs (DeBose-Boyd et al. 1998; Zheng et al. 2002; Paschinger et al. 2004, 2005), core 1 O-glycan T-synthase (Ju et al. 2006), and several glycosaminoglycan glycosyltransferases (Berninsone et al. 2001; Herman et al. 1999; Herman and Horvitz 1999; Hwang et al. 2003). A search of the C. elegans genome revealed at least 24 different putative 1,2FTs, 5 1,3FTs, and 1 1,6FT, but their biological functions in the worm remain unknown.
The value of C. elegans as a model for exploring the importance of glycosylation in development was the discovery of the eight sqv (squashed vulva) mutants that affect vulval invagination (Herman et al. 1999). A loss of the sqv gene function can cause a severe reduction in the hermaphrodite fertility. The eggs produced by these mutants were arrested during embryogenesis, suggesting that they are required maternally during development. Molecular analysis of the sqv genes revealed them to be enzymes involved in the proteoglycan biosynthetic pathway (Herman and Horvitz 1999). The sqv-7 gene encodes a nucleotide sugar transporter that can translocate UDP–glucuronic acid, UDP–N-acetylgalactosamine, and UDP–galactose (Berninsone et al. 2001). The sqv-3 and sqv-8 have been identified as genes encoding a Gal I- and GlcA I-transferase, respectively (Bulik et al. 2000), whereas sqv-2 and sqv-6 encode a Gal II and Xyl-transferase, respectively (Hwang et al. 2003).
A number of recent studies have provided structural information on C. elegans glycans showing the existence of fucose-rich N-glycans with both core and terminal fucose (Altmann et al. 2001; Guerardel et al. 2001; Cipollo et al. 2002; Haslam et al. 2002; Natsuka et al. 2002; Schachter et al. 2002; Haslam and Dell 2003; Cipollo et al. 2005). The N-glycans of C. elegans have recently been shown to be unique at each developmental stage, indicating that glycoconjugate biosynthesis may be involved during development (Cipollo et al. 2005). The large number of putative FTs and the lack of sialylation make C. elegans an ideal model for studying FTs. The identification and characterization of each member of the 1,3FT family is the first step in understanding their functions and roles in development.
Here, we report on the identification, molecular cloning, characterization, and comparison of five 1,3FTs from C. elegans (CEFT). The enzymes designated CEFT-1, CEFT-2, CEFT-3, CEFT-4, and CEFT-5 are type II membrane proteins, but they exhibit limited identity to each other. Except for CEFT-5, each member of the CEFTs is distinguished by acceptor specificity, cation requirement, and sensitivity to temperature. The expression pattern of CEFT-1 by promoter analysis shows that expression is limited to a few cells. The results show that each member of the 1,3FT family has unique acceptor specificity and expression in C. elegans, which could reflect their various biological roles.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Identification of
1,3FTs in C. elegansOur previous studies identified both
1,2FT and 1,3FT activities from extracts of C. elegans (DeBose-Boyd et al. 1998; Zheng et al. 2002). The first 1,3FT, termed CEFT-1, was identified by Basic Local Alignment Search Tool search of the C. elegans genome using a conserved region of mouse and human fucosyltransferase 4 (FUT4) (DeBose-Boyd et al. 1998). Four other putative 1,3FTs with homology to CEFT-1 were identified from the C. elegans database (Supplemental Figure 1). They contain the conserved 1,3FT motif (FxL/VxFENS/TXXXXYxTEK) from sequence alignment of all the cloned 1,3FTs (Martin et al. 1997; Breton et al. 1998). Two putative 1,3FTs (CEFT-2 and CEFT-3) were contained in cosmid T05A7 (GenBank Accession # U40028
[GenBank]
) and encode a 378 and a 392 amino acid polypeptide, respectively (Supplemental Figure 1B and C). A fourth putative 1,3FT (CEFT-4) was contained in cosmid F59E12 (GenBank Accession #AF003386) and encodes a 400 amino acid polypeptide (Supplemental Figure 1D). A fifth putative 1,3FT (CEFT-5) was contained in cosmid K12H6 (GenBank Accession # AC006674
[GenBank]
) and encodes a 382 amino acid polypeptide (Supplemental Figure 1E). The family shares 25–31% identity with mammalian 1,3FTs (Breton et al. 1998). The genes of the C. elegans 1,3FT family are located in chromosome II, suggesting that these FT genes may have evolved by successive duplications. The genomic structures of the CEFTs have multiple exons (7–10) and introns (6–9), in contrast with the six human 1,3-FT/1,4-FT that have one exon each (Supplemental Figure 2).
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Kyte–Doolittle hydrophilicity plots (Kyte and Doolittle 1982
) of the CEFTs protein sequences predict transmembrane domains at the N-termini typical of most Golgi-localized glycosyltransferases (Joziasse 1992; Kleene and Berger 1993) (Supplemental Figure 3). The CEFTs contain one to six potential N-glycosylation sites (Figure 2A), and the alignment of the CEFTs shows sequence identity of approximately 23% (Figure 1).
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Cloning of the CEFT complementary deoxyribonucleic acid cDNAs from C. elegans
Gene-specific primers were used to amplify the entire coding sequence of each CEFT from a C. elegans cDNA
ZAP library, as described in the Materials and methods section. A CEFT-1 plasmid construct encoding a 433 amino acid protein was generated (Supplemental Figure 1A). This construct encodes a protein with a shorter N-terminal region than the previously published protein sequence of this enzyme (DeBose-Boyd et al. 1998). This difference may be due to a different splicing, or misinterpretation of the coding sequence.
Expression of C. elegans CEFT cDNAs in COS7 cells
To determine whether the C. elegans sequences encode functional 1,3FTs, we constructed plasmids encoding the full-length protein of each CEFT with an N-terminal human protein C-4 (HPC4)-epitope tag (Figure 2A). Recombinant enzymes were made by transient transfection of the CEFT plasmid constructs into COS7 cells. Three days post-transfection, the expression of the recombinant enzymes was confirmed by western blotting with the HPC4-Ab, identifying recombinant proteins of the expected sizes that were variably glycosylated. The recombinant HPC4 epitope-tagged CEFTs were purified by absorption onto HPC4-conjugated beads. The presence of possible N-glycans on the enzymes was examined by treatment with peptide N-glycosidase F (PNGaseF), which reduced their apparent sizes, indicating that each enzyme was N-glycosylated (Figure 2B).
Acceptor specificity of recombinant CEFTs
To characterize the putative FT activities of the CEFTs, the recombinant enzymes were purified from the medium using HPC4-conjugated beads and tested with different acceptors under a variety of conditions (Table I). Previously, CEFT-1 was shown to have an extremely weak activity toward the acceptor lacto-N-neotetrose (LNnT) (DeBose-Boyd et al. 1998). However, we found that CEFT-1 expressed a much higher activity toward the N-glycan pentasaccharide core acceptor Man1-3(Man1-6)Manß1-4GlcNAcß1-4GlcNAcß1-Asn (M3) with or without the 1,6-linked fucose (M3F6). The enzyme did not act on the core pentasaccharide if it was elongated (Table I), which confirms the recent findings of Paschinger et al. (2004); in their study on this same enzyme that they designated FUT-1. The products generated by recombinant CEFT-1 were examined by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy-TOF-MS) (MALDI) to confirm the addition of one fucose (Figure 3A). We also tested the activity of the enzyme toward the acceptor Man1-3(Man1-6)Manß1-4GlcNAcß1-4(Fuc1-6)GlcNAcß1-Asn (M3F6), which resulted in the addition of a second fucose residue. Analysis of this product and the acceptor M3F6 by 1H-nuclear magnetic resonance (NMR) showed that the addition of this second fucose residue resulted in the appearance in the spectrum of doublets for H-1 at 
= 5.124/5.134 and CH3 protons at d = 1.291 ppm, which are essentially similar to values reported previously for a Fuc in -anomeric configuration at C-3 of GlcNAc-1 of the core (Staudacher et al. 1991). Furthermore, introduction of the second Fuc resulted in an upfield shift in the resonance of the NAc–CH3 of GlcNAc-1 and GlcNAc-2, and a downfield shift in the resonance of the H-1 of the 1,6-linked Fuc. On the basis of the earlier reported 1H-NMR data of these structures, we propose that the product is the difucosylated Man1-3(Man1-6)Manß1-4GlcNAcß1-4(Fuc1-6)(Fuc1-3)GlcNAcß1-Asn or M3F6F3 (Staudacher et al. 1991). Thus, the results demonstrate that CEFT-1 is a core 1,3FT catalyzing the transfer of fucose to the innermost GlcNAc of the N-glycan acceptors M3 or M3F6.
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In contrast, CEFT-2 transfers fucose more efficiently to LDNT (GalNAcß1-4GlcNAcß1-3Galß1-4Glc), and less efficiently to LN-pNP (Galß1-4GlcNAc
-pNP) and LNnT (Galß1-4GlcNAcß1-3Galß1-4Glc) (Table I). The specificity of this enzyme to LacdiNAc (LDN) is novel and notable, because most N-glycans in helminths are built upon this type of precursor disaccharide (Kang et al. 1993; Van den Eijnden et al. 1997). Both CEFT-3 and CEFT-4 transfer fucose to LN-pNP and LNnT acceptors (Table I). While both CEFT-3 and CEFT-4 are highly active toward the LN-pNP acceptor, CEFT-4 activity toward LNnT is minimal (10%), demonstrating the distinct acceptor specificity of each enzyme. CEFT-5, however, did not have any activity toward any acceptor in our panel. This may be due to an inactive enzyme, or the absence of the right acceptor in the assays.
To confirm the nature of the linkages in the products of these various CEFTs, the 3H-fucosylated products from each enzyme reaction were treated with either 1,2-fucosidase, 1,3/4-fucosidase, or 1,6-fucosidase, and the radiolabeled release was measured. Treatment of the products with 1,3/4-fucosidase released over 90% of the 3H–fucose compared with minimal release upon treatment with either 1,2- or 1,6-fucosidase, demonstrating that the linkages are 1,3; the 1,4 is precluded because the C-4 position in the acceptor is occupied. The products of these CEFTs were also analyzed by Dionex to determine their structures in comparison with known standards (Figure 3B), and the results confirmed in each case the product as shown. Thus, each of the putative FTs, except for CEFT-5, is an 1,3FT although each has different optimal acceptor requirements.
Temperature and cation requirements of the recombinant CEFTs
Further analyses were conducted to find the optimum conditions for each enzyme. Activity assays were carried out for 1 h either at room temperature, approximately 23 °C, or at 37 °C. Surprisingly, the activities of the CEFT enzymes were reduced by >80% at 37 °C compared with room temperature (Table III). The cation requirement assays for the CEFTs were performed at room temperature using a variety of metals at 20 mM (Table III). CEFT-1 prefers Mn2 + for optimum activity, whereas in the presence of Cu2+ and Zn2+ at 1 mM concentrations the enzyme was inactive. CEFT-2 prefers Ca2+ for optimum activity, whereas Cu2+ and Ni2+ greatly reduced its activity. CEFT-3 acts optimally in the presence of ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid (EDTA/EGTA), indicating that cations inhibit this activity. CEFT-4 prefers no additional metals or Ca2 +, whereas Ni2+ greatly reduced its activity. Ca2+ (10 mM) appeared optimal for CEFT-4 activity. Interestingly, the enzymes (CEFT-1, CEFT-2) that require cations for optimum activity were the most sensitive to elevated temperature.
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Expression of CEFTs messenger ribonuclic acid (mRNA) at various stages in C. elegans development
To examine the CEFTs expression during development, we analyzed their transcript levels by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Two different sets of primers were used for each CEFT to control for primer variability. Equivalent amounts of total RNA from different developmental stages including L1, mixed L2–4, and young adult were analyzed (Figure 4). The results indicate that the CEFT genes are expressed at all developmental stages with little relative a difference in expression when normalized to
-actin.
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Expression of the CEFT-1 promoter at various stages in C. elegans development
C. elegans gene expression patterns are generally determined by generating a fusion of the promoter of the gene of interest to a reporter such as lacZ or green fluorescent protein (GFP) (Fire et al. 1990
; Chalfie et al. 1994). The reporter gene can provide information on temporal and spatial distribution of the gene. To localize the expression of the CEFT-1 gene, we used reporter–promoter constructs driving the expression of the GFP in the vector pPD95.67/CEFT-1-prom (see Materials and methods). A 1521-bp promoter sequence upstream to CEFT-1 was ligated into the GFP reporter vector and co-injected with the pBX injection marker into pha-1 worms. The expression of the GFP reporter gene in transgenic worms injected with the CEFT-1 promoter construct pPD95.67/CEFT-1-prom was examined (Figure 5).
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GFP expression was specifically localized to a few cells at the anterior and posterior regions of the worm. At the anterior region, the GFP expression was identified in two amphid neurons (ASG), the pharyngeal intestinal (PI) valve (six cells), one to two neurons in retrovesicular ganglion (RVG), and two neurons posterior to the PI valve. The GFP expression in the posterior region was localized to the two phasmid neurons (PHA, PHB) and the anal valve (four cells). The amphid and phasmid neurons were identified using DiI stain. RVG and posterior neurons were identified using a neuron-specific Ab (goat anti-unc18/rabbit-anti-GFP) and dipeptidyl aminopeptidase I for deoxyribonucle (DNA) identification. The results indicated that the promoter of CEFT-1 is specifically activated in chemosensory neurons (amphids and phasmid neurons) and valve regions (PI valve and anal valve). Promoter expression was evident through all developmental stages with a highly restricted GFP expression.
Mutagenesis of the CEFT genes
To explore the developmental functions of these enzymes, mutant worms containing large deletions in the CEFT genes were obtained from the two C. elegans Reverse Genetics Core Facilities in Oklahoma City, OK and Vancouver, Canada. The centers are part of the International C. elegans Gene Knockout Consortium. The nonspecific deletions by mutagens (TMP/UV or EMS) generated mutant worms with only partial sequences of the CEFT genes (Supplemental Figure 2). The deleted regions were validated by performing PCR across each deletion site. The mutant worms were designated CEFT-1/VC378, CEFT-2/RB511, CEFT-3/RB706, CEFT-4/VC182, and CEFT-5/VC212 and had a deletion of 597, 1876, 839, 497, and 554 bp, respectively. The mutant strains were back-crossed 6 times with normal animals to insure that any phenotype observed is due to the CEFT allele deletion. The large deleted region in each strain would abolish the enzyme function due to non-expression, truncation, or codon frame shift.
Phenotypic analysis of C. elegans expressing mutated CEFT genes
The mutant worms were screened for physical and behavioral abnormalities. All five mutants have normal morphology and movement indistinguishable from wild-type N2 worms. Phenotypic screening for differences in life span (12 ± 2 days), brood size (225–265), and defecation intervals (45 ± 2 s) also failed to identify a clear difference from wild-type N2 worms. Mutant worm extracts were assayed for loss of activity with specific acceptors. The only difference detected by these assays was between wild-type and CEFT-1 mutant worms. The CEFT-1 mutant completely lost its core fucosylating activity (Figure 6A). A western blot of CEFT-1 mutant extract using an antibody (A6b) to horseradish peroxidase, which cross-reacts with glycans expressing the core 1-3-fuc (Paschinger et al. 2004), did not detect significant levels of glycoprotein antigens (Figure 6B).
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These results, using a different mutant of C. elegans compared with the studies of Paschinger et al. (2004)
, confirm that CEFT-1 is responsible for synthesizing the glycans expressing the 1-3-fucosylated core structures in N-glycans. These results demonstrate that none of the individual CEFTs are required for worm development. |
Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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The results of our study demonstrate that each of the
1,3FTs (CEFT-1 through CEFT-4) exhibits unique acceptor specificity and differs in other properties such as cation requirements. Interestingly, all of these enzymes that exhibited activity were inactivated at 37 °C, and maximal activity was observed at room temperature range. Paschinger et al. (2004) have reported that the C. elegans core 1,6FT activity was less active at 37 °C compared with room temperature. Warren et al. (2002) also reported temperature and ion sensitivity for the C. elegans gly-2 gene that is similar to the mammalian GlcNAc-TV. This phenomenon may be common to glycosyltransferases from organisms such as C. elegans that live at environmental temperatures.
Previous studies had indicated that CEFT-1 recognized lactosamine-containing glycans (DeBose-Boyd et al. 1998), although the activity was extremely low. The results of our study here show that in fact CEFT-1 exhibits profound specificity and very high activity for pauci-mannose-type N-glycans containing three mannosyl residues in the presence or absence of the core 1,6-Fuc. These results are consistent with the recent study of Paschinger et al. (2004), who also reported that CEFT-1 exhibited activity toward the pauci-mannose acceptor and that a deletion of the CEFT-1 gene causes loss of the core 1,3FT activity in worm extracts. Our results here extend these previous findings, since we now report the relative activities of CEFT-1 to other 1,3-CEFTs and have defined their unique acceptor specificities. In addition, we have found that expression of CEFT-1 is extremely limited and promoter analysis shows that expression is primarily localized to a few cells at the worms anterior and posterior regions. The restricted expression is comparable with the immunostaining pattern seen with the core 1,3-Fucose cross-reactive horseradish antibodies in permeabilized C. elegans (Siddiqui and Culotti 1991). The highly specific expression to a few cells is similar to that reported for one of the C. elegans 1,2-FTs (Zheng et al. 2002). The highly regulated regional expression of individual glycosyltransferases in C. elegans may account for the presence of a remarkably high number of FT genes in this organism compared with vertebrates.
A number of 1,3FT activities have been identified and cloned from plants and invertebrates, which are similar to CEFT-1 in their ability to generate an 1-3-fucosylated core structure in N-glycans (Wilson and Altmann 1998; Wilson et al. 1998, 2001; van Tetering et al. 1999; Bakker et al. 2001; Fabini et al. 2001; Wilson 2001). N-glycans containing the core 1,3-fucose have been implicated in allergy and immunogenic reactions because of the interaction of the hosts with allergens or helminthes (van Die et al. 1999; van Ree et al. 2000; Faveeuw et al. 2003; van Die and Cummings 2006). In Haemonchus contortus, fucosylated LacdiNAc (LDNF) antigens had been identified earlier as a potential protective glycan component in the host–parasite interaction (Vervelde et al. 2003). A more recent study (Geldhof et al. 2005) suggests that the protective component possibly may also include core 1-3-fucosylated N-glycan structures.
CEFT-2 fucosylates LDN to make the LDNF structure, but can also fucosylate LNnT to generate LDNFIII. Both CEFT-3 and CEFT-4 specifically fucosylate the acceptor structure LNnT. This shows that C. elegans CEFTs are able to catalyze the synthesis of the LDNF and Lewisx determinants. LDN-specific monoclonal Ab studies and mass spectrometry analysis of C. elegans glycans have detected LDN-based structures, which are common to helminthic glycoconjugates (Nyame, et al. 1999; Haslam and Dell 2003). In helminths, the Lewisx antigen has been found in schistosomes (Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium), but not in C. elegans, by enzyme-linked immunosorbent assay test and western blot analysis using monoclonal Abs (Nyame et al. 1998). This does not rule out the possibility that C. elegans does make Lewisx-related structures in a context that is not recognized by the restricted Ab to the Lewisx antigen. For example, studies of C. elegans glycans have found unusual structures with fucose linked to galactose (Haslam and Dell 2003). The Lewisx epitope, if capped by other sugars, would not likely be recognized by the anti-Lewisx Ab. Additionally, most of the studies of C. elegans glycans have used glycosidases to release the N-glycans, but such enzymes may not quantitatively release all the N-glycans, especially those with unusual core modifications. Further characterizations of the C. elegans, N-glycan structures are needed to identify the specific substrate for each CEFT. The CEFT-5 inactivity may be resolved if its possible substrate can be predicted by such glycan analyses.
Toward characterizing the biological roles of CEFTs, deletion mutants were successfully generated for each CEFT. The mutant animals, however, developed normally, suggesting that the individual enzymes are not essential for development. The CEFT-1 deletion mutant worm has a complete loss of activity resulting in the absence of a core 1,3-fucose on N-glycans. This demonstrates that C. elegans has only one core 1,3-FT and is consistent with the fact that the other members of the family utilize other acceptors. The core 1-3-fucosyl linkages may have other functions in C. elegans that remain to be discovered through more novel studies on phenotypes. For example, earlier studies on the functions of complex-type N-glycans in C. elegans were explored through null mutations of the three GNT-I genes, but no obvious phenotypes were revealed at that time (Zhu et al. 2004), although more recent studies show that expression of the GNT-I genes is associated with innate immunity and resistance to bacterial pathogens (Shi et al. 2006). In addition, recent studies indicate that triple-null mutants for these genes in Drosophila are associated with defects in locomotion and a reduced life-span (Sarkar et al. 2006). With regard to the other CEFTs, the ability of CEFT-2, CEFT-3, and CEFT-4 to fucosylate the same LNnT acceptor, although weakly, may allow them to compensate for each other, and this might explain the normal phenotype of the individual deletion mutants. Future studies on the CEFT knockouts should be directed to defining their glycomes to reveal if loss of activity correlates with loss of structure or if some type of compensation is occurring.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Materials
All the chemicals and reagents used in this study, unless otherwise indicated, were from Sigma Chemical Co. (St. Louis, MO). The C. elegans cDNA library was a gift from Dr Robert Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK). FuGene 6 and complete protease inhibitor mixture were from Roche Molecular Biochemicals (Indianapolis, IN). N-glycanase was from Glyko (Novato, CA). Carbograph was purchased from Alltech (Deerfield, IL). Sep-Pak C18 cartridges were purchased from Waters Corp (Milford, MA). Taq DNA polymerase and other PCR components were obtained form Boehringer Mannheim (Indianapolis, IN). Oligonucleotide primers were synthesized by Invitrogen (Carlsbad, CA). The Eukaryotic TA Cloning Kit (bidirectional) was obtained from Invitrogen. Restriction enzymes were purchased from New England Biolabs, Inc. (Beverly, MA). GDP-[3H]Fuc was purchased from NEN Life Science Products (Boston, MA).
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). Lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose III were obtained from V-labs, Inc. (Covington, LA). The tetrasaccharide LDNT, GalNAcß1-4GlcNAcß1-3Galß1-4Glc, was synthesized in our laboratory by the addition of a terminal GalNAc residues to LNT-2 upon incubation with C. elegans ß1,4–GalNAcT and UDPGalNAc (Kawar et al. 2002). The Galß1-4GlcNAc-pNP (LN-pNP), GlcNAcß1-4GlcNAc (chitobiose), GlcNAcß1-4GlcNAcß1-4GlcNAcß1-4GlcNAcß1-4GlcNAcß1-4GlcNAcß1-4 (chitohexaose) were purchased from Toronto Research Chemicals Inc. (Downsview, Ontario, Canada). Bovine immunoglobulinG (IgG) was purchased from Equitech-Bio Inc. (Kerrville, TX) to make the GnM3F6, M3F6, and M3 acceptors in our laboratory. Bovine IgG treated overnight with Pronase was purified and treated with ß-galactosidase to make the GnM3F6 acceptor. Additional treatment with hexosaminosidase generated the M3F6 acceptor, and the M3 acceptor was subsequently made by treatment with 1,6-fucosidase.
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 nematode growth medium plates seeded with OP50 bacteria. The mixed-stage worms were washed with M9 buffer (22 mM KH2PO4, 22 mM Na2HPO4, 85 mM NaCl, 1 mM MgSO4, add 10 volumes of basic hypochlorite (0.25 M KOH, 1–1.5% hypochlorite, freshly mixed) 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 of CEFTs
Specific forward and reverse oligonucleotide primers for each CEFT were used to amplify the coding sequence from a C. elegans cDNA library in the ZAP vector (provided by Dr Robert Barstead). CEFT-1 forward primer 5'-TCCACCATGACTGCAAGAAGCATCAAACTTTTCTTTGCAAGATGGAAATATTTAATGTTTGCTTGT-3' and reverse primer 5' ATCTAACGGAATAGAATCTACTAGTGTTCC-3'; CEFT-2 forward primer 5'-GCCGCCACCATGAAACATAA-TACTTTACGAGCC-3' and reverse primer 5'-TAAAAACC-GAGTTGCAAACGAATTATC-3'; CEFT-3 forward primer 5'-GCCGCCACCATGTCTCAAATAGGCGGTGCCA-3' and reverse primer 5'-CACTGGAGCGCAAATATTTCGAAGCT-ATCTGATTATTGC-3'; CEFT-4 forward primer 5'-GCC-GCCACCATGAGGGTTCGGCCAGCAAGTGTC-3' and reverse primer 5'-ACCACTGAGCCAACTGCGC-3'; CEFT-5 forward primer 5'-GCCGCCACCATGTCTTCGACAAGT-GGCAATTTCTG-3' and reverse primer 5'-TATCAAATAC-TTCATTCCAATTGAATTATTACACAC-3'. PCR reaction mixture contained 2.5 U of Taq DNA polymerase, 10 µM of each primer, 200 µM dNTPs, 2.5 mM MgCl2, and 2 µL of diluted (10 fold) C. elegans ZAP vector in a final volume of 50 µL. For CEFT-2 and CEFT-3, a first round of PCR was carried out using T7 primer to amplify low copy templates and a 40-fold dilution was used as templates for amplification. Amplification was carried out by an initial denaturing step of 94 °C for 5 min, followed by 35 cycles of 94 °C/1 min, 52 °C/1 min, and 68 °C/2.5 min. These cycles were then followed by extension period of 72 °C/10 min. Following amplification, the PCR product was run on an agarose gel and the single DNA band was excised from agarose gel and purified by QIAquick Gel Extraction Kit (QIAGEN, Inc. San Clarita, CA). The purified DNA was inserted into the pCR3.1 vector (Bidirectional Eukaryotic TA Cloning Kit) by ligation and subsequently transformed into One Shot Top10F' Invitrogen competent cells according to the procedures provided with the kit. Clones containing inserts were selected and amplified, and plasmids were isolated using QIAGEN Kits and digested with restriction enzyme to check the direction of the inserts. The clones with inserts in the right orientation were sequenced by facilities available at the Oklahoma Medical Research Foundation, and plasmids were prepared using QIAGEN Endotoxin-Free Plasmid DNA Purification Kit.
Construction and expression of N-terminal HPC4 epitope-tagged CEFTs
N-terminal HPC4 epitope-tagged CEFTs were constructed using PCR for introduction the 12 amino acid Ca2 +-dependent HPC4 epitope (EDQVDPRLIDGK) (Stearns et al. 1988; Rezaie et al. 1992) into the cDNAs. CEFT-1 forward primer 5'-TCCACCATGGAGGACCAGGTGGACCCCAGGC-TGATCGACGGCAAGATGACTGCAAGAAGCATCAAA-3' and reverse primer 5'-CTAATCTAAC-GGAATAGAATCT-AC-3'; CEFT-2 forward primer 5'-TCCACCATGGAGGA-CCAGGTGGACCCCAGGCTGATCGACGGCAAGATGAA-ACATAATACTTTACGA-3' and reverse primer 5'-TCATAAAAACCGAGTTGCAAACGA-3'; CEFT-3 forward primer 5'-TCCACCATGGAGGACCAGGTGGACCCCAGG-CTGATCGACGGCAAGATGTCTCAAATAGGCGGTGCC-3' and reverse primer 5'-CTACAAATATTTCGAAGCTATC-TG-3'; CEFT-4 forward primer 5'-TCCACCATGGAGGAC-CAGGTGGACCCCAGGCTGATCGACGGCAAGATGAGGGTTCG GCCAGCAAGT-3' and reverse primer 5'-CTAAC-CACTGAGCCAACTGCGCAC-3'; CEFT-5 forward primer 5'-TCCACCATGGAGGACCAGGTGGACCCCAGGCTGA-TCGACGGCAAGATGTCTTCGACAAGTGGCAAT-3' and reverse primer 5'-CTATATCAAATACTTCATTCCAAT-3' Using plasmid pCR3.1/CEFTs cDNA as the template, the PCR was performed by denaturation at 94 °C for 5 min, followed by 25 cycles at 94 °C/1 min, 55 °C/1 min, 68 °C/2.5 min, and an extension period of 72 °C/10 min. The PCR product was purified and TA cloned as described in Cloning of CEFTs.
Expression of CEFTs in COS7 cells
To determine whether the cloned insert encoded an active 1,3FT, COS7 cells were transfected with 4.5 µg of plasmid pCR3.1 harboring cDNA encoding each of the CEFTs using FuGene 6 Transfection Reagent, as described by the manufacturer (Boehringer Mannheim). Mock-transfected cells were prepared by transfecting COS7 cells with pCR3.1 plasmid without insert. Three days after transfection, the transfected cells were harvested, suspended in 50 mM sodium cacodylate buffer containing 1 tablet/10 mL of Complete Protease Inhibitor Cocktail (Boehringer Mannheim), 1% Triton X-100 and then subjected to sonication (three pulses of 10 s each, setting 5) on a cell disruptor (Branson SONIFIER Cell Disruptor 185, Branson Sonic Power Co., Danburg, CT). The cell lysate was then incubated on ice for 30 min to allow for the solubilization of proteins, and then centrifuged at 2000 g at 4 °C for 10 min. The supernatant fraction was collected and either used directly for FT assays, or stored as aliquots at –80 °C. Frozen extracts were thawed only once for use in enzyme assays.
Preparation of HPC4–UltraLinkTM medium
Forty milligrams of HPC4 monoclonal Ab was dissolved in 20 mL of 0.1 M 3-morpholinopropane sulfonic acid and 0.6 M sodium citrate, pH 7.5, and coupled to 0.6 g of UltraLinkTM Bio-support medium (Pierce Biotechnology, Rockford, IL) 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 recombinant HPC4-tagged CEFTs on HPC4-UltraLinkTM Bio-support medium beads
To capture the recombinant HPC4-tagged CEFTs on HPC4-UltraLink Bio-support Medium beads, the cell extracts containing recombinant HPC4-tagged CEFTs containing 1 mM CaCl2 were incubated with HPC4-UltraLinkTM beads at 4 °C for 4 h. The beads were collected by centrifugation at 2 000g for 5 min and washed 3 times with 50 mM Tris–HCl, pH 7.2, 1 M NaCl, and 1 mM CaCl2. The beads were then washed 2 times with 50 mM Tris–HCl, pH 7.2, 150 mM NaCl, and 1 mM CaCl2 and assayed for FT activity as described in FT assays.
FT assays
The FT assays were performed in 50 µL reaction mixtures containing 100 mM sodium cacodylate (pH 7.0), 20 mM cation (MnCl2, CaCl2, etc.) or 10 mM EDTA/EGTA, 5 mM ATP, 15 mM Fuc, 50µM GDP-[3H]-Fuc (40–50,000 cpm/nmol), 1 mM acceptor substrate and 10µL of cell extract or HPC4-UltraLinkTM beads. Reaction mixtures were incubated at room temperature or 37 °C for 1–5 h and terminated by the addition of 450 µL water. The neutral reaction products were isolated by ion exchange chromatography on 0.5 mL columns of QAE-Sephadex (Sigma-Aldrich) to separate unincorporated label. If the acceptors were glycopeptides or oligosaccharides, the product was separated by chromatography on a 1-mL Carbograph column (Alltech, IL). For those oligosaccharide acceptors with a hydrophobic aglycon (pNP), the product was isolated by Sep-Pak C18 cartridges (Waters, Milford, MA). The isolated radioactive product was assayed by liquid scintillation.
Characterization of FT products
The product obtained form CEFT-1 with M3 and cold GDP-Fuc was analyzed by mass spectrometry. MALDI-TOF-MS was carried out on a Voyager-DE RP Biospectrometry Workstation instrument (Applied Biosystems, Framingham, MA) equipment with a pulsed nitrogen laser (337 nm). MALDI spectra were acquired at 20 and 25 kV accelerating voltage in positive or negative ion modes, respectively, whereas the low-mass gate was used to discard the ions with m/z values of <400. All acquired spectra were smoothed by applying a 19-point Savitzky-Golay smoothing routine. The matrix used for positive mode was 10 mg/mL 2,5-dihydroxybenzoic acid prepared in 50% acetonitrile and 0.1% trifluoroacetic acid. For the negative mode, 10 mM ammonium citrate solution was used. The sample spots were air dried. The instrument was calibrated with a ladder of hyaluronic acid-derived glycans. Average masses of the [M + H]+ or [M + Na]+ (positive mode) or [M + H]– (negative mode) ions were calculated according to EXPASy GlycoMod Tool and Glycan Mass (http://us.expasy.org/tools/glycomod/). All masses were generally within 0.1–0.5 mass units of the calculated values. Products of CEFT-2, CEFT-3, and CEFT-4 with their acceptors (LDNT, LNnT) were analyzed by HPAEC on a CarboPacTM PA1 column (4 x 250 mm) (Dionex Corporation, Sunnyvale, CA), using an isocratic 20 mM NaOH fluid solution. Further analysis of the radiolabeled products, generated by recombinant CEFTs with their preferred acceptors and GDP–[3H]fucose donor was carried out by treating them with either H2O (mock) or 0.8 mU 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. Following treatment, the samples were isolated as described above and the radioactivity in the eluted material determined by scintillation counting. NMR analysis was performed as described previously (Kawar et al. 2002).
Preparation of DNA constructs for promoter analysis
To examine the spatial and temporal pattern of CEFTs expression during C. elegans development, we prepared fusion constructs of the upstream promoter sequences of CEFT-1 fused to the GFP. A 3781-bp fragment of genomic sequence containing the putative promoter region immediately upstream of initiation site for CEFT-1 translation was obtained by PCR using C. elegans genomic DNA as template with primers, 5'-GGTTCTAGGTGCTAAAAATGCGG-3', and 5'-CGTCTGTAAATCGAGCAGCATG-3'. The PCR product was treated with XbaI/HindIII restriction enzymes to generate a 1521-bp fragment. The reaction was run on an agarose gel and the DNA band was excised from agarose gel and purified by QIAquick Gel Extraction Kit. The purified DNA was ligated into the MCS of pPD95.67 vector that was also digested with XbaI/HindIII restriction enzymes (originally provided by Dr Andrew Z. Fire, Carnegie Institute of Washington, Baltimore, MD) creating plasmid pPD95.67/CEFT-1-prom.
Preparation of transgenic worms and promoter analysis
DNA injection into the C. elegans germ line was carried out as described in Mello and Fire (1995) and Mello et al. (1991). Transgenic lines were established by co-injection with 100 ng/µL of CEFT-1::GFP constructs (pPD95.67/CEFT-1-prom) and pBX (100 ng/µl), which carries pha-1(e2123) gene to serves as a transformation marker. Postinjection, the worms were grown at 14 °C for 4 days followed by 4 days at 25 °C. Transformants were selected by selecting animals at the L2 stage.
Preparing synchronous culture of L1, L2–L4, and young adult worms
The L1, L2–L4, and young adult stages of N2 worms were prepared as described previously (Zheng et al. 2002).
Quantitative real-time RT-PCR analysis of C. elegans CEFT mRNA during development
Quantitative RT-PCR was performed using an ABI Prism 7700 sequence detection (PerkinElmer Life and Analytical Sciences, Waltham, MA) using the double-stranded DNA binding dye, SYBR Green. Random primed first strand cDNAs were prepared from the total RNA from staged synchronous populations as described in Zheng et al. (2002). The first strand cDNA was used as a template in PCR reactions to amplify cDNAs encoding CEFT transcripts using two set of primers for each CEFT in reactions containing 3 µL of first strand cDNA in a volume of 50 µL. CEFT-1 forward primer(1) 5'-TTTGTCCCGAAGCCTAATCAA-3' and reverse primer(1) 5'-TGATATAATCAGGCCTTGGAATTTG-3'; CEFT-1 forward primer(2) 5'-AACAGCTTCATTGCTATTGACGAT-3' and reverse primer(2) 5'-CCATGTAGGCGGTCTTATTATTCAT-3': CEFT-2 forward primer(1) 5'-ATGTGCCAGCTGAAGTGAACTACA-3' and reverse primer(1) 5'-TTGTTCTGTGCATTTCCATTTGA-3'; CEFT-2 forward primer(2) 5'-TTGTGCATCATATCCAAAATGTGA-3' and reverse primer(2) 5'-TTGGTATCCACAATTTTGAATTGTTT-3': CEFT-3 forward primer(1) 5'-CGGCTGGTGCACACTTTG-3' and reverse primer(1) 5'-TGAATGCCACCATGCAACAT-3'; CEFT-3 forward primer(2) 5'-AGATTATCGTGGCAGTACGGATAAAT-3' and reverse primer(2) 5'-CGGACTTTCGAGAGACCATAGC-3': CEFT-4 forward primer(1) 5'-AGCTTCTCTGGAAAGCGAAAGA-3' and reverse primer(1) 5'-TCGCATTGAGATTCGTTGTCA-3'; CEFT-4 forward primer(2) 5'-TGCATCCGATGATCGACTTAAAT-3' and reverse primer(2) 5'-CTCGATCGCAATGTAGAATGGA-3': CEFT-5 forward primer(1) 5'-TGGAAAAGGTCGGGAGTGAA-3' and reverse primer(1) 5'-CCTGAGAACCCGTTGTCGAATA-3'; CEFT-5 forward primer(2) 5'-CGGGAAAATGATCCAGAAAGG-3' and reverse primer(2) 5'-CGTCCGTTTTTGTACTCCCATAC-3' 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 in the identical conditions described for the CEFTs. Cycling parameters were 95 °C for 15 s and 60 °C for 1 min. To confirm the absence of non-specific amplification, the PCR products were analyzed by 1.2% agarose gel containing 10 µg/mL ethidium bromide.
Peptide:N-glycosidase F (PNGase F) digestion of recombinant of CEFTs
The HPC4-tagged CEFTs purified from the HPC4 affinity column were treated with 1/U of PNGase F (PerkinElmer Life and Analytical Sciences) at 37 °C for 24 h. After the treatment, the reaction mixture was run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4–20%) and analyzed by western blot with HPC4 monoclonal Ab.
Mutagenesis of the CEFTs gene
The CEFT-1/VC378, CEFT-4/VC182, and CEFT-5/VC212C mutant strains were prepared by the Reverse Genetics Core Facility in Vancouver, Canada, and the CEFT-2/RB511 and CEFT-3/RB706 strains were prepared by the Reverse Genetics Core Facility in Oklahoma City, OK. The centers are part of the International C. elegans Gene Knockout Consortium. The five CEFT strains were a gift from the Knockout Consortium. The strains received were not out-crossed and the flanking regions around the breakpoints are known, but the exact coordinates are ambiguous. The deleted region was confirmed by performing PCR across the deleted site. Two sets of primers (nested primers) were generated for each strain. The outer set was used for performing PCR across the deleted site and the inner primers were used for sequencing the PCR product. CEFT-1 forward primer(OL) 5'-ACACATGAAAAAACAAAACAC-3' and reverse primer(OR) 5'-TCACAAACCAAGCAGCTCC-3'; CEFT-1 forward primer(IL) 5'-ATGGAAATATTTAATGTTT-3' and reverse primer(IR) 5'-GCCGAAATCCCAGTGTCATGT-3': CEFT-2 forward primer(OL) 5'-CCACTCCATTTTTCGATGT-3' and reverse primer(OR) 5'-CTCGGATAGTATAACCTGGAAGA-3'; CEFT-2 forward primer(IL) 5'-CGGGATATCAATGAGACG-3' and reverse primer(IR) 5'-CTTCCCATTGATCGTAACTCCTTC-3': CEFT-3 forward primer(OL) 5'-TGTGCCGAAAAGTTGTTTGT-3' and reverse primer(OR) 5'-CAAATATTTCGAAGCTATCTG-3'; CEFT-3 forward primer(IL) 5'-AACTGATTCCGATGTCTGGGACC-3' and reverse primer(IR) 5'-CACATTTCGAATGAATGC-3': CEFT-4 forward primer(OL) 5'-GCGGGCGTTTTTGGAAATG-3' and reverse primer(OR) 5'-CAAAACGACCACTATTATCAGGGG-3'; CEFT-4 forward primer(IL) 5'-TCAAAGTCGGAGATGTTG-3' and reverse primer(IR) 5'-GTATCAATTACACAACGATC-3': CEFT-5 forward primer(OL) 5'-TCGTCATATATTCCAACTAGC-3' and reverse primer(OR) 5'-GAGTTGACGTCGATTTTGG-3'; CEFT-5 forward primer(IL) 5'-GCAGAAGGTCACGGGTTCAAT-3' and reverse primer(IR) 5'-ACCTGCCTGTGACCTACCTACAAC-3'. Amplification was carried out by an initial denaturing step of 94 °C for 5 min, followed by 35 cycles of 92, 55 °C/1 min, and 72 °C/2.5 min. These cycles were then followed by an extension period of 72 °C/10 min. The PCR products were purified with QIAquick PCR Purification Kit and sequenced using the inner primers (IL and IR). The strains were back-crossed 6 times to insure that any phenotype observed is due to the deletion of a specific CEFT. Standard back-crossing were done as follows: strains were back-crossed to normal males, male progeny were selected and back-crossed to normal hermaphrodites, male progeny were selected and screened by PCR for the deletion, and mated to normal hermaphrodites. Heterozygous progeny were identified by PCR and its progeny were screened for homozygous mutants.
Phenotypic analysis of the mutant strains
The life span of the mutant strains was determined by monitoring 40 single eggs after egg-laying (time zero) until they no longer moved when prodded. The animals were transferred onto fresh plates daily during the egg laying stage. Brood size was determined by monitoring a single L4 larvae and counting its progeny at the L3/L4 stage. Defecation intervals were monitored by timing the interval of muscular contractions of defecation at the posterior end. Ten animals were scored for 10 defecation cycles and averaged.
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/)
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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None declared.
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Footnotes |
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2 Present address: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd NE Suite #4001, Atlanta, GA 30322
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Abbreviations |
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Ab, antibody; CEFT, Caenorhabditis elegans
1,3-fucosyltransferase; cDNA, complementary deoxyribonucleic acid; DNA, deoxyribonucleic acid; EDTA/EGTA, ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FT, fucosyltransferase; Fuc, (1-3,4)fucosidase; FUT4, fucosyltransferase 4; GalNAc, N-acetylgalactosamine; GDP–fuc, guanosine 5'-diphosphate-(1-3,4) fucosidase; GFP, green fluorescent protein; GlcNAc, N-acetylglucosamine; HPC4-Ab, human protein C IV-antibody; IgG, immunoglobulin G; LDN, LacdiNAc; LDNF, fucosylated LacdiNAc; LDNT, GalNAcß1-4GlcNAcß1-3Galß1-4Glc; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy; mRNA, messenger ribonucleic acid; NMR, nuclear magnetic resonance; PI, pharyngeal intestinal; PNGaseF, peptide: N-glycosidase F; RT-PCR, reverse transcriptase-polymerase chain reaction; RVG, retrovesicular ganglion. |
References |
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