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Molecular cloning and characterization of an [alpha]1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Molecular cloning and characterization of an [alpha]1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans
Introduction
Terminal Fuc[alpha]1->3GlcNAc linkages are critical components in the molecular events underlying a variety of biological phenomena, such as the adhesion of activated leukocytes to inflamed endothelium, embryogenesis, immune responses, and cancer metastasis (Kannagi, 1997; Kim and Varki, 1997; McEver and Cummings, 1997; Staudacher, 1997). These fucosylated determinants, occurring in the Lewis x (Lex) and sialyl Lex antigens, are displayed in a tissue and/or cell-type specific manner, due in part to the restricted expression of the glycosyltransferases, especially the [alpha]1,3 fucosyltransferases ([alpha]1,3FT) involved in their biosynthesis (Schachter, 1994). Several [alpha]1,3FTs have been identified and characterized from both vertebrates, invertebrates and in prokaryotes and molecular cloning has revealed an emerging family of [alpha]1,3FTs (Goelz et al., 1990; Kukowska-Latallo et al., 1990; Kumar et al., 1991; Lowe et al., 1991; De Vries andVan Den Eijnden, 1992; Weston et al., 1992a,b; Natsuka and Lowe, 1994; Natsuka et al., 1994; Sasaki et al., 1994; Chan et al., 1995; Gersten et al., 1995; DeBose-Boyd et al., 1996; Lee et al., 1996; Mulder et al., 1996; Smith et al., 1996; Ge et al., 1997; Martin et al., 1997; Oulmouden et al., 1997; Sajdel-Sulkowska et al., 1997).
Our interest in [alpha]1,3 fucosylated glycans and [alpha]1,3FTs stems from observations that many of the helminthic parasites, such as Schistosoma mansoni, Dirofilaria immitis, and Haemonchus contortus synthesize antigenic protein- and lipid-associated glycoconjugates in which Fuc[alpha]1->3GlcNAc is a major determinant (Ko et al., 1990; Srivatsan et al., 1992a,b; Kang et al., 1993; Cummings and Nyame, 1996; Richter et al., 1996). Interestingly, some of these antigenic glycans are high molecular weight N-glycans containing polylactosamine [->3Gal[beta]1->4GlcNAc[beta]1->]n in which most of the inner GlcNAc residues in the repeating units are [alpha]1,3 fucosylated and the elongated glycans are capped by the Lex antigen, Gal[beta]1->4[Fuc[alpha]1->3]GlcNAc, at the nonreducing terminus. Some of the N- and O-glycans in both S. mansoni and D. immitis also contain fucosylated lacdiNAc (LDN) structures GalNAc[beta]1->4[Fuc[alpha]1->3]GlcNAc (Srivatsan et al., 1992a; Kang et al., 1993). It has been proposed that the immune responses generated toward fucosylated glycans may play a role in the pathogenesis of schistosomiasis (Velupillai and Harn, 1994; El Ridi et al., 1996).
Recently, we identified an [alpha]1,3FT in crude extracts of adult schistosomes and adult H. contortus that can synthesize [alpha]1,3 fucosylated glycans (R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations; DeBose-Boyd et al., 1996). The [alpha]1,3FTs display enzymatic properties that closely resemble those of human FTIV, which is expressed in myeloid cells. These studies led us to survey other helminths for the expression of an [alpha]1,3FT and/or the expression of Lex glycoconjugates. We now report our finding that the free-living nematode Caenorhabditis elegans expresses an [alpha]1,3FT activity capable of synthesizing Lex-containing glycoconjugates in vitro. Characterization of the enzymatic properties of the C.elegans [alpha]1,3FT reveal it to be similar in many enzymatic properties to the [alpha]1,3FT expressed by H.contortus and schistosomes. A search of the C.elegans genome database revealed a candidate gene, named CEFT-1, which has sequence similarities to known [alpha]1,3FTs. The cDNA was cloned and expression of the recombinant enzyme reveals that it is an [alpha]1,3FT and can promote Lex, but not sialyl Lex, expression in transfected COS7 cells. Although much is known about the enzymology and molecular biology of fucosylation, much remains unclear about the potential functions of fucosylated glycans in development and cellular differentiation. The discovery of fucosylation pathways in C.elegans and the power of these worms as an experimental system for biological studies may provide further insights into the possible biological functions of fucosylated glycans.
Results
Identification of an [alpha]1,3FT activity in total C.elegans homogenates
We and others have reported that Schistosoma mansoni, a parasitic helminth in animals and humans, is able to synthesize Lex antigens (Ko et al., 1990; Srivatsan et al., 1992b; van Dam et al., 1994). Based on this observation, we initiated a survey of other helminths for the presence of Lex determinants. The results of our studies thus far indicate that Lex expression is restricted to schistosomes, but interestingly, Fuc[alpha]1->3GlcNAc linkages may be common among all helminths (A. K. Nyame, R. A. DeBose-Boyd, T. D. Long, and R. D. Cummings, unpublished observations). For example, D.immitis and H.contortus lack Lex antigen, but can synthesize Fuc[alpha]1->3GlcNAc linkages(R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations; Kang et al., 1993; Haslam et al., 1996). During this survey, we found that the free-living nematode, C.elegans, like most other helminths, lacked expression of either the Lex or sialyl Lex antigens. However, to determine whether C.elegans contains an [alpha]1,3FT activity, detergent homogenates of adult worms were incubated with the acceptor oligosaccharide LNnT in the presence of Mn2+ and the sugar nucleotide donor, GDP-[3H]-Fuc. Analyses of the reaction products demonstrated that extracts of C.elegans contain a fucosyltransferase activity capable of generating a 3H-Fuc-labeled product from LNnT in a manner dependent upon time and the amount of protein in the assay (Figure
Figure 1. Fucosyltransferase activity in C.elegans homogenates. Detergent extracts of adult C.elegans were prepared in the presence of protease inhibitors and reacted with 25 µM GDP-3H-Fuc and 5 mM LNnT in 100 mM sodium cacodylate buffer, pH, 7.0 containing 20 mM MnCl2, 15 mM fucose, and 5 mM ATP. Following incubation at 25°C, reactions were applied to columns of QAE-Sephadex and analyzed as described in Materials and methods. (A) Fucosyltransferase activity in C.elegans extracts (100 µg) with respect to time. (B)Fucosyltransferase activity in C.elegans extracts with respect to protein in a 4 h assay.
The product was isolated and analyzed by Dionex HPAEC, where it coeluted with standard LNFPIII (Figure
Although [alpha]1,3FTs share significant sequence similarities at the amino acid level, each [alpha]1,3FT displays distinct enzymatic characteristics, such as acceptor specificity (Natsuka and Lowe, 1994). To further characterize the [alpha]1,3FT activity in C.elegans extracts, [alpha]1,3FT assays were performed with a panel of glycan acceptors. As shown in Figure
Figure 3. Acceptor specificity of CEFT-1. (A) Assays of the [alpha]1,3FT present in C.elegans homogenates and (B) recombinant CEFT-1 expressed in transfected COS7 cells. Fucosyltransferase assays were conducted as described in the Materials and methods using 5 mM of LNnT, LNT, 2,3sLN, 2,6sLN, and LDNT as acceptors. The results are expressed as relative activity, where the activity obtained with the acceptor LNnT is taken as 100%. Identification of a fucosyltransferase gene in C.elegans cosmid K08F8 Members of the emerging [alpha]1,3FT family exhibit a high degree of sequence similarity at the amino acid level. To identify C.elegans sequences with possible homologies to known fucosyltransferases, the sequences of human and mouse FTIV were used to perform a TBLASTN search of the C.elegans genome data base. This search resulted in the identification of the C.elegans cosmid KO8F8 (GenBank accession #Z66497), which harbors a gene with significant sequence similarity to human and mouse FTIV (Wilson et al., 1994). The product of the putative C.elegans [alpha]1,3FT is a 1353 bp cDNA (named CEFT-1) which is predicted to encode a 451 amino acid polypeptide (Figure
The Kyte-Doolittle hydrophilicity plot (Kyte and Doolittle, 1982) of the protein sequence suggests the presence of a 22 amino acid transmembrane domain at the N-terminus (Figure Cloning of the cDNA from C.elegans encoding CEFT-1
Gene-specific primers were designed to amplify the entire coding sequence of the C.elegans CEFT-1 from a C.elegans cDNA [lambda]ZAP library, as described in Materials and methods. Following amplification, the PCR product was TA-cloned into the mammalian expression vector pCR3.1. A clone was isolated and DNA sequencing confirmed the exon/intron boundaries predicted by the database. However, the cloned cDNA contained 78 base pairs of the first intron, beginning at nucleotide 90 and ending at nucleotide 167 (Figure Southern and Northern blot analysis
Mammalian tissues are known to express a wide variety of [alpha]1,3FTs in a tissue- and/or cell type-specific manner. To test the possibility that C.elegans also express additional [alpha]1,3FTs with sequence similarity to CEFT-1, C.elegans genomic DNA was analyzed by Southern blot, using restriction enzymes that are not predicted to cleave within the introns or exons of the CEFT-1 gene. A single major band was seen for all digests, consistent with the presence of a single copy gene (Figure
In Northern analysis the CEFT-1 cDNA hybridizes to a major 1.9 kb transcript in total C.elegans RNA preparations; faint hybridization was observed to another band at ~4 kb (Figure
To determine whether the intron-containing transcript of CEFT-1 (+ partial intron 1) with the additional 78 bp intronic sequence exist in adult C.elegans, we performed both RT-PCR analysis and genomic PCR. Primers were designed to produce either a 104 bp product from transcripts that lack the 78 bp partial intronic sequence, or a 182 bp product from those containing the partial intronic sequence. Transcripts containing the complete intron 1 with 335 bp would generate a product containing 438 bp total. All PCR products were treated with the restriction enzyme BspE1, which is predicted to have a cleavage site within the 78 bp partial intronic sequence and at the same position in the 335 bp complete intron 1, but there are no other BspE1 sites within the gene. Total RNA was isolated and oligo dT-primed cDNA was used as a template to amplify the CEFT-1 cDNA. Control incubations used plasmids containing either the intron-containing clone and a continuous ORF. RT-PCR of the two control plasmids with and without the intronic sequence led to the formation of products corresponding to 182 bp and 104 bp, respectively, as predicted (Figure
Figure 7. RT-PCR and genomic PCR of CEFT-1 from adult C.elegans. Total C.elegans RNA was used as templates in first strand cDNA synthesis. An aliquot of the cDNA synthesis reaction was used as a template to amplify CEFT-1 coding sequences with primers and conditions, as described in the Materials and methods (lanes 7 and 8). As controls the cDNAs encoding CEFT-1 (+ partial intron 1) (lanes 1 and 2), CEFT-1 (- partial intron 1) (lanes 3 and 4), and C.elegans genomic DNA (lanes 5 and 6) were also amplified. Following amplification, samples were either subjected to treatment with BspEI (lanes 1, 3, 5, and 7) or no treatment (lanes 2, 4, 6, and 8), and subsequently fractionated on a 2% agarose gel containing ethidium bromide and analyzed. Size markers in bp are indicated. Demonstration that CEFT-1 encodes a functional [alpha]1,3FT
To confirm that the cDNA encoding CEFT-1 corresponds to an [alpha]1,3-FT activity, the cDNA encoding CEFT-1 (± partial intron 1) was transfected into the African green monkey kidney cell line COS7. COS7 cells do not contain endogenous [alpha]1,3FT activity; however, these cells synthesize the type-1 and -2 oligosaccharide precursors, which are suitable substrates for [alpha]1,4 and [alpha]1,3 fucosylation, respectively (Kukowska-Latallo et al., 1990; Saski et al., 1994). Transfection of COS7 cells with cDNA encoding CEFT-1 (- partial intron 1) containing a continuous ORF results in about 20-fold level of activity over control cells, using LNnT as the acceptor (Table I). When cells were transfected with the cDNA encoding CEFT-1 (+ partial intron 1) containing the 78 base pair intronic sequence, significant enzyme activity above control levels was obtained (Table I). This level was about 1/4 of that obtained with the cDNA containing a continuous ORF. These results confirm that CEFT-1 encodes a functional [alpha]1,3FT. The lower level of activity obtained with CEFT-1 (+ partial intron 1) compared to the cDNA lacking this segment, may reflect the inefficiency of splicing this mRNA in mammalian cells.
Table I.
To confirm the fucosyl linkages in the reaction product generated by CEFT-1, the 3H-Fuc-labeled product was isolated and subjected to treatment with the [alpha]1,3/4 fucosidase followed by descending paper chromatography. Treatment of the product with the [alpha]1,3/4 fucosidase caused quantitative release of 3H-fucose from the radiolabeled pentasaccharide product (Figure
Figure 8. Characterization of the reaction product generated by recombinant CEFT-1. The radiolabeled product obtained with COS7 cell extracts transiently expressing CEFT-1 were assayed using LNnT as the acceptor and GDP-[3H]-Fuc as the donor. The isolated product was analyzed by descending paper chromatography before (open circles) and after treatment (solid circles) with the Streptomyces [alpha]1,3/4 fucosidase. Arrows indicated the migration positions of the standard LNFPIII and free fucose. Further characterization of recombinant CEFT-1
To further characterize the CEFT-1 [alpha]1,3FT activity, the acceptor specificity of the enzyme was assessed. Fucosyltransferase assays were performed using CEFT-1-transfected COS7 cell extract and a variety of oligosaccharide acceptors. The recombinant CEFT-1 has a clear preference for nonsialylated type-2 acceptors over either neutral type-1 acceptors or sialylated type-2 acceptors (Figure
We next questioned whether the recombinant CEFT-1 in the transfected COS7 cells acts on endogenous glycoproteins. Extracts of CEFT-1-transfected COS7 cells were analyzed by ELISA, using mAb specific for Lex (CD15) or sLex (CSLEX-1) antigens. As controls, we utilized extracts from transfected COS7 cells expressing human FTIV. We observed reactivity of CEFT-1-transfected cells with CD15, indicating expression of Lex, as well as FTIV-transfected cells (Figure
Figure 9. Reactivity of CEFT-1-transfected COS7 cells with anti-Lex antibodies. Extracts of COS7 cells transfected with cDNA encoding (A) human FTIV, (B) CEFT-1, and (C) mock transfected cells were homogenized and extracts coated onto microtiter wells at indicated protein concentrations. Following blocking with 1% BSA/PBS, expression of Lex was monitored by reactivity with CD15, as described in Materials and methods. Assays were performed in triplicate and standard deviations are shown by the bars. Data obtained with complete assay components including the primary mAb CD15 (open circles); controls in which the primary mAb CD15 was omitted (solid circles).
These studies demonstrate that the soil nematode C.elegans expresses an [alpha]1,3FT (CEFT-1) capable of acting on both LN-containing acceptor glycans to generate the Lex antigen, and on LDN-based acceptor glycans to generate [alpha]1,3 fucosylated LDN-based structures. The catalytic activity of recombinant CEFT-1 is highly similar to human FTIV. Both enzymes act predominantly on type-2 rather than type-1 acceptor glycans and neither are efficient with [alpha]2,3-sialylated type-2 acceptors (Natsuka and Lowe, 1994; Staudacher, 1996). In addition, both enzymes, when expressed in COS7 cells cause formation of the Lex, but not sialyl Lex antigen.
Although CEFT-1 can generate the Lex antigen in vitro with appropriate acceptors, we have been unable to detect the presence of Lex or sialyl Lex in extracts of total adult C.elegans (A.K.Nyame, R.A.DeBose-Boyd, T.D.Long, and R.D.Cummings, unpublished observations). However, C.elegans contains abundant levels of fucosylated glycans reactive with Lotus tetragonolobus agglutinin (LTA; A.K.Nyame, R.A.DeBose-Boyd, T.D.Long, and R.D.Cummings, unpublished observations). We recently demonstrated that LTA binds with high affinity to nonsialylated glycans containing either terminal Lex structures or the LDNF-motif GalNAc[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->R (Yan et al., 1997). Our preliminary studies also indicate the existence of a [beta]1,4-N-acetylgalactosaminyltransferase ([beta]1,4GalNAcT) in extracts of adult C.elegans, like the enzyme in schistosomes (Srivatsan et al., 1994), that can synthesize the LDN-based structures. In H.contortus, an intestinal parasitic helminth in sheep, we found expression of a [beta]1,4GalNAcT and an [alpha]1,3FT, similar to CEFT-1 in C.elegans, but H.contortus lack expression of a [beta]1,4GalT capable of synthesizing LN-based structures (R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations). Structural determinations of N- and O-glycans in glycoproteins from C.elegans and H. contortus are in progress, and these structural definitions should provide important insights into the likely biosynthetic pathways operative in these organisms.
Interestingly, genomic sequencing in C.elegans has identified a gene related to mammalian [beta]1,4GalT (Wilson et al., 1994). Whether the enzyme encoded by this putative [beta]1,4GalT is actually capable of transferring Gal from UDPGal, or utilizes other sugar nucleotide donors, such as UDPGlcNAc, as seen for other members of the [beta]1,4GalT family (Bakker et al., 1994), remains to be determined. It is conceivable that glycans containing the LN-motif and Lex-based structures are synthesized by C.elegans, but other capping reactions or masking may occur that abolish reactivity with anti-Lex antibodies, as seen, for example, in murine teratocarcinoma cells (Cho et al., 1996). It will be important in future studies to carry out detailed structural analyses of the glycans synthesized by all stages of C.elegans and many other helminths to resolve these interesting possibilities.
Interestingly, intron 1 of the CEFT-1 gene has two tandem introns with two pairs of donor/acceptor slice sites. This unusual tandem intron may not be efficiently spliced since the major cDNA identified for CEFT-1 in this study contains 78 bp of intron 1. This partial intron would not be expected to allow generation of a functional enzyme, since it contains a stop codon. This partial intron must be a substrate for splicing reactions in vivo, since transfection of COS7 cells with CEFT-1 (+ partial intron-1) cDNAs leads to [alpha]1,3FT activity; however, enzyme levels are very low relative to that observed for CEFT-1 (- partial intron 1). Although such tandem introns as we observed for CEFT-1 are unusual, there is some evidence for unusual intron splicing in other organisms. Several genes of Euglena gracilis chloroplasts contain twintrons, i.e., introns-within-introns, and undergo stepwise splicing reactions leading to functional protein products (Copertino et al., 1992; Hallick, 1992).
Results presented in Figure
The sequence of CEFT-1 displays a relatively low identity (~23%) to the sequences of vertebrate [alpha]1,3FT, such as human FTIII and bovine FT, but the identity in the predicted catalytic C-terminal domain is significantly higher. CEFT-1 contains the conserved motif identified for several vertebrate and prokaryotic [alpha]1,3FTs (H.pylori) (YxFxL/VxFENSxxxxYxTEK; Figure
C.elegans homogenates, in addition to an [alpha]1,3 FT, probably contain several other fucosyltransferases. We found that extracts contain an [alpha]1,2FT that can generate the H-type antigen LNFPI, using the type-1 acceptor LNT, whereas this [alpha]1,2FT activity does not act on the type-2 acceptor LNnT. Two types of [alpha]1,2 fucosyltransferases have been identified in humans, the H-blood group fucosyltransferase and the Secretor fucosyltransferase (Larsen et al., 1990; Kelly et al., 1995). Both enzymes transfer Fuc in the [alpha]1,2 linkage to terminal Gal residues on both type-1 and -2 backgrounds. Further studies are required to determine if the C.elegans [alpha]1,2 FT is homologous to [alpha]1,2 FTs in other systems or if it represents a novel enzyme.
We also found that extracts of C.elegans contain a fucosyltransferase that acts on an [alpha]2,3-sialylated acceptor to generate sialyl Lex, whereas the recombinant form of CEFT-1 acts poorly on such acceptors (Figure
Over the past several years, [alpha]1,3 fucosylation has received attention due in part to the role played by glycans containing Fuc[alpha]1->3GlcNAc linkages in cell adhesion, as seen in selectin functions with leukocytes and endothelium and cell-cell interactions during development (Staudacher, 1996; Kim and Varki, 1997; McEver, 1997; McEver and Cummings, 1997). Fucosylated glycans are often expressed in a restricted manner in development, differentiation, and progression of metastases (Schachter, 1994). This restricted expression implies that these glycans have importance in development, but the developmental roles of fucosylated glycans remain elusive. The ease of genetic manipulation of C.elegans, their small genome, the detailed knowledge of cell lineage, and the ease with which the organisms can be cultivated, make these organisms a valuable model system to study the importance of fucosylation in animal biology. Materials
Sodium cacodylate, MnCl2, ATP, l-fucose, GDP-fucose, and QAE-Sephadex were purchased from Sigma Chemical Co. (St. Louis, MO). Lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFPI), lacto-N-fucopentaose II (LNFPII), lacto-N-fucopentaose V (LNFPV), 2,3-sialyl-N-acetyllactosamine (2,3sLN), and 2,6-sialyl-N-acetyllactosamine (2,6sLN) were obtained from V-Labs, Inc. (Covington, LA). The tetrasaccharide LDNT GalNAc[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc was synthesized in our laboratory by degalactosylation of LNnT with jack bean [beta]-galactosidase (Sigma) and addition of terminal GalNAc residues upon incubation with bovine [beta]1,4 galactosyltransferase (Sigma) and UDPGalNAc (Chiu et al., 1994). GDP-[3H]-Fuc (7.1 Ci/mmol), 32P-dCTP (3000 Ci/mmol), and GeneScreen Plus hybridization transfer membranes were purchased from NEN Life Science Products (Boston, MA). Streptomyces [alpha]1,3/4 fucosidase (1 mU/ml) was acquired from Pan Vera Corporation (Madison, WI). Monoclonal antibody specific for Lex (anti-CD15) determinants was obtained from Becton-Dickinson (San Jose, CA). Anti-Lea antibodies were acquired from Signet Laboratories, Inc. (Dedham, MA). Anti-mouse IgM- and IgG-peroxidase conjugates were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Anti-sLex mAb (CSLEX-1) was derived from hybridomas obtained from ATCC. Oligonucleotide primers were synthesized by Operon Technologies, Inc. (Alameda, CA). Taq DNA polymerase and other PCR components were obtained from Boehringer Mannheim (Indianapolis, IN). The Eukaryotic TA Cloning Kit (bidirectional) was obtained from Invitrogen (San Diego, CA). Restriction enzymes were purchased from New England Biolabs, Inc. (Beverly, MA). Preparation of C.elegans extracts
Frozen C.elegans adults were a kind gift of Dr. James B. Rand of the Oklahoma Medical Research Foundation. Worms were suspended in 50 mM cacodylate buffer, pH 7.0, containing 1 tablet/10 ml of Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Indianapolis, IN), homogenized on ice with a Biohomogenizer (model M 133/1281-0; Biospec Products, Inc., Bartlesville, OK) using three pulses of 10 s each, and subsequently subjected to sonication (three pulses of 10 s each) on a Branson Cell Disrupter (model 185) to effect complete disruption of the worms. The homogenate was adjusted to 1% Triton X-100 and incubated on ice for 30 min to allow solubilization of proteins. The homogenate was centrifuged at 14,000 r.p.m., at 4°C, for 10 min, and the supernatant fraction was collected. Protein concentration of the extract was determined by the BCA protein assay procedure (Pierce Co., Rockford, IL). Detergent solubilized extracts were either used directly or stored as aliquots at -80°C. Frozen extracts were thawed only once for use in enzyme assays. Fucosyltransferase assays
Fucosyltransferase assays were performed in 50 µl of 100 mM sodium cacodylate buffer, pH 7.0, containing 20 mM MnCl2, 5 mM ATP, 15 mM fucose, 25-50 µM GDP-[3H]-Fuc (50-65,000 c.p.m./nmol) as the sugar-nucleotide donor, 5 mM LNnT, LNT, LDNT, 2,3sLN, or 2,6sLN as acceptors and detergent solubilized extracts of C.elegans adults or CEFT-1- or FTIV-transfected COS7 cells, or parental COS7 cells as the source of fucosyltransferase. Reactions were incubated for different time intervals and terminated by the addition of 450 µl water for reactions containing the neutral acceptors, LNnT, LNT, or LDNT, or terminated by addition of 450 µl pyridine/acetate buffer for reactions using either 2,3sLN or 2,6sLN acceptors. C.elegans extracts were assayed at 25°C, but assays with COS7 cell extracts were conducted at 37°C. Neutral products were isolated by ion exchange chromatography on 0.5 ml columns of QAE-Sephadex equilibrated in water. The columns were washed with deionized water and 0.5 ml fractions were collected. Radioactivity associated with each fraction was determined by liquid scintillation counting. Products of the assays employing 2,3sLN or 2,6sLN were isolated by applying the reaction mixtures to 0.5 ml columns of QAE-Sephadex equilibrated with 2 mM pyridine/acetate buffer, pH 5.5. The columns were washed with 2.5 ml of 2 mM pyridine/acetate to remove, unbound, neutral material. Bound material, containing the charged product, was subsequently eluted from the column by washing with 2.5 ml of 25 mM pyridine/acetate buffer and collecting 0.5 ml fractions. For each enzyme assay, control experiments were performed without added acceptor. Radioactivity obtained in these control assays represented free fucose and was subtracted as background. All assays were performed in duplicate and results reported are an average with an overall standard error of less than 5%. Characterization of fucosyltransferase assay products
Products obtained with LNnT and either extracts of C.elegans or COS7 cells expressing recombinant CEFT were isolated by ion-exchange chromatography and descending paper chromatography. The isolated, radiolabeled products were incubated with 25 µU of Streptomyces [alpha]1,3/4 in 25 µl of 50 mM KH2PO4, pH 6.0, containing 0.1 M NaCl and 0.02% NaN3 for 24 h at 37°C. Following treatment, the samples were analyzed by descending paper chromatography in a solvent system of ethyl acetate/pyridine/acetic acid/water (5:5:1:3). In addition, products obtained with LNT and 2,3sLN were characterized by high-pH anion exchange chromatography (HPAEC) using a Carbopak PA1 column (4 × 250 mm) on a Dionex instrument. The samples were separated isocratically at 100 mM NaOH for 30 min at a flow rate of 1 ml/min. Fractions were collected at 30 sec intervals, and radioactivity in each fraction was determined by liquid scintillation counting. Cloning and expression of CEFT-1
The oligonucleotides, 5[prime]-CTCAAAT GCCTTTCCACC-3[prime] and 5[prime]-CAACTAATCTAACGGAATAGAATC-3[prime] were used as forward and reverse primers, respectively, to amplify the CEFT-1 coding sequence from a C.elegans cDNA library in the [lambda]ZAP vector, kindly provided by Dr. Robert Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK). Reaction mixtures for amplification by Taq DNA polymerase contained 0.2 µM of each primer, 200 µM dNTPs, 2 mM MgCl2, and 5 µl of 109 pfu/ml C.elegans adult cDNA library in a final volume of 100 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, 53°C/1 min, and 72°C/1 min. These cycles were then followed by extension period of 72°C/7 min. Following amplification, an aliquot of the [ap]1.4 kbp product was analyzed on an agarose gel, and the remainder of the product was ligated into the pCR3.1 vector and subsequently transformed into One Shot TOP10F' competent cells according to procedures provided with the Bidirectional Eucaryotic TA Cloning Kit. Clones containing either sense or antisense inserts were selected, amplified, and maxipreps were prepared using Qiagen kits (QIAGEN, Inc., San Clarita, CA). Sequencing of several clones was performed by the Oklahoma State University Recombinant DNA/Protein Resource Facility (Stillwater, OK).
To determine if the cloned inserts encoded an active [alpha]1,3FT, COS7 cells were transfected with 5 µg of plasmid DNA harboring either CEFT-1 (± partial intron 1) (in the plasmid pCR3.1) or FTIV cDNA (in the plasmid pRC/RSV) using lipofectamine reagent, as described by the manufacturer (GIBCO BRL, Gaithersburg, MD). Three days posttransfection, cells were harvested, solubilized in 50 mM sodium cacodylate buffer containing 1% Triton X-100, and assayed for fucosyltransferase activity as described above. Mutagenesis of CEFT-1(+ partial intron 1) cDNA
Mutagenesis of the cloned cDNA encoding CEFT-1 (+ partial intron 1) was carried out to eliminate the 78 bp of intron 1. Overlapping fragments of 109 and 1281 bp generated in primary PCR reactions were used as templates in a secondary PCR reaction to generate the intron-free cDNA encoding CEFT-1 (- partial intron 1). The 109 bp fragment was synthesized using 5[prime]-TTACTGACAACATGAAAAAACAAAACACTC-3[prime] (forward primer) and 5[prime]-ATATTTCCATAAAAAGCGAAGGAATCTGTC-3[prime] (reverse primer). The 1281 bp fragment was generated using 5[prime]-TTCCTTCGCTTTTTATGGAAATATTTAATGTTTGC-3[prime] (forward primer) and 5`-ACTAATCTAACGGAATAGAATCTACTAGTGT-3[prime] (reverse primer). Using 1 ng of the cloned CEFT-1 (+ partial intron 1) cDNA as a template, the two overlapping fragments were generated in reactions containing 2.5 U of Taq DNA Polymerase, 0.250 µM of each primer, 200 µM dNTPs, and 3 mM MgCl2 in 100 µl. After an initial denaturing step of 94°C for 5 min, denaturation, annealing, and extension were carried out sequentially at 94°C/1 min, 65°C/1 min, and 72°C/2 min, respectively, for 35 cycles. Specifically amplified products of 109 and 1281 bp were then isolated from agarose gels and subsequently used as templates in a third PCR reaction. The two overlapping fragments were then incubated in PCR reactions containing 2.5 U of Taq DNA polymerase, 200 µM dNTPs, and 3 mM MgCl2 to allow the formation of a heteroduplex between the two, overlapping fragments. The heteroduplex formed was extended by Taq DNA polymerase for 1 cycle which included a denaturation step of 94°C for 1 min, an annealing step of 62°C/1 min, and an extension period at 72°C/10 min. Following the extension of the heteroduplex into a full-length product, amplification was achieved by including 0.2 µM of the oligonucleotides, 5[prime]-ACAACATGAAAAAACAAAACACTCCTC-3[prime] (forward primer) and 5[prime]-CTAATCTAACGGAATAGAATCTACTAG-3[prime] (reverse primer). Amplification was carried out using the same program for generating the 109 and 1281 bp products described above. RT-PCR and genomic PCR of CEFT-1
The existence of a fully processed cDNA encoding CEFT-1 (- partial intron 1) was demonstrated using RT-PCR in conjunction with restriction enzyme digestion. Oligo-dT primed first strand cDNA was synthesized, utilizing Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD) under recommended conditions,from 6 µg of total C.elegans RNA, isolated using the RNAgents Total RNA Isolation System (Promega, Madison, WI). The first strand cDNA was used as a template in PCR reactions to amplify cDNAs encoding CEFT-1 transcripts using the oligonucleotides, 5[prime]-AACCAGTTGTCGTTGGCTC-3[prime] (forward primer) and 5[prime]-GAGCATAAATAACAAGAAGATATGTG-3[prime] (reverse primer) in reactions containing 10 µM of each primer, 2.5 U of Taq DNA polymerase, 2.5 mM MgCl2, 200 µM dNTPs, and 2 µl of first strand cDNA or 100 ng of C.elegans genomic DNA in a volume of 100 µl. After an initial annealing step of 94°C for 5 min, the reactions were subjected to 35 cycles of 94°C 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a final extension at 72°C for 7 min. In control experiments, the same primer pair was used to amplify the cDNAs encoding CEFT-1 (± partial intron 1) from 1 ng of the plasmids harboring each of the inserts, using identical conditions described. Following PCR amplifications, amplified fragments were concentrated by precipitation and subsequently treated with 20 U of the restriction enzyme BspEI for 1 h at 37°C. Following enzyme digestion, reactions were fractionated on a 2% agarose gel containing 10 µg/ml ethidium bromide. ELISA
Cell extracts from COS7 cells transfected with CEFT-1 (± partial intron 1) and FTIV cDNAs and nontransfected cells were diluted in PBS at various concentrations, and absorbed onto wells of microtiter plates for 2 h at room temperature. Following coating, wells were blocked with a 5% BSA/PBS solution and were washed 5× in PBS/0.3% Tween-20 solution. Monoclonal antibodies reactive with Lex, Lea, and sLex determinants were incubated with coated wells for 1 h at room temperature. After a 5× washing step, bound primary antibodies weredetected using horseradish peroxidase-conjugated goat anti-mouse IgM antibodies. Following addition of substrate, absorbance was read at 405 nm. Southern and Northern blots
High molecular weight C.elegans genomic DNA was prepared from adult worms using procedures described previously (at Website htpp://eatworms.swmed.edu). Genomic DNA (10 µg) was digested with restriction endonucleases, fractionated through a 0.9% agarose gel, and subjected to Southern blot analysis. Briefly, restricted DNA was transferred to nylon membranes and cross-linked by UV irradiation. Blots were prehybridized in Rapid Hyb hybridization buffer (Amersham Life Science) according to the manufacturer's procedures and subsequently hybridized in the same buffer containing CEFT-1 (+ partial intron 1) cDNA radiolabeled with 32P-dCTP by random priming (Pharmacia Biotech). After a 2 h hybridization period, blots were subjected to two 15 min low stringency washes (2 × SSC, 0.1% SDS) at room temperature, followed by a 30 min, high stringency wash at 65°C in 0.1 × SSC, 0.1% SDS. The blots were then subjected to autoradiography.
For Northern blots, total RNA was prepared from C.elegans adults using the Total RNA isolation kit (Promega). RNA samples were electrophoresed through a 1.2% agarose gel containing formaldehyde and transferred to a nylon membrane. Northern blots were prehybridized in Rapid Hyb buffer as described by the manufacturer's procedures, and probed with the radiolabeled CEFT-1 (+ partial intron 1) cDNA probe. Following hybridization, blots were rinsed twice at room temperature with 2 × SSC, 0.1% SDS and stringently washed at 65°C with 0.1 × SSC, 0.1% SDS. The blots were then exposed to film and processed.
This work was supported by NIH Grant AI26725 to R.D.C. and a National Research Service Award GM18274-02 from the NIGMS to RDB. We thank Dr. Robert Barstead and Dr. James Rand for providing C.elegans adult cDNA library and C.elegans adults, and Kim Ballard for excellent technical assistance.
Lex, Lewis x antigen; sLex, sialyl Lewis x antigen; HPAEC, high pH anion exchange chromatography; FT, fucosyltransferase; CEFT-1, C.elegans fucosyltransferase-1; LNT, lacto-N-tetraose, Gal[beta]1->3GlcNAc[beta]1->3Gal[beta]1->4Glc; LNnT, lacto-N-neotetraose, Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc; 2,3sLN, 2,3-sialyl-N-acetyllactosamine, NeuAc[alpha]2->3Gal[beta]1->4GlcNAc; 2,6sLN, 2,6-sialyl-N-acetyllactosamine, NeuAc[alpha]2->6Gal[beta]1->4GlcNAc; LDN, lacdiNAc, GalNAc[beta]1->4GlcNAc; LDNF, [alpha]1,3 fucosylated LDN, GalNAc[beta]1->4(Fuc[alpha]1->3)GlcNAc; LDNT, lacdiNAc-tetraose, GalNAc[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc; LNFPIII, lacto-N-fucopentaoseIII, Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1-> 3Gal[beta]1->4Glc; LNFPII, lacto-N-fucopentaoseII, Gal[beta]1->3Fuc[alpha]1->4)GlcNAc[beta]1->3Gal[beta]1->4Glc; LNFPI, lacto-N-fucopentaoseI, Fuc[alpha]1->2Gal[beta]1->3GlcNAc[beta]1->3Gal[beta]1->4Glc; LNFPV, lacto-N-fucopentaoseV, Gal[beta]1,3GlcNAc[beta]1,3Gal[beta]1,4(Fuc[alpha]1,3)Glc.
Figure 2. Characterization of C.elegans fucosyltransferase products obtained with the acceptors LNnT and LNT. (A) The product of C.elegans fucosyltransferase with the acceptor LNnT was isolated and analyzed by Dionex HPAEC, as described in Materials and methods. Arrows indicate the elution positions of standards. (B) The product of C.elegans fucosyltransferase with the acceptor LNnT was isolated and analyzed by descending paper chromatography before (open circles) and after treatment (solid circles) with the Streptomyces [alpha]1,3/4 fucosidase. Arrows indicated the migration positions of the standard LNFPIII and free fucose. (C) The product of C.elegans fucosyltransferase with the acceptor LNT was isolated and analyzed by Dionex HPAEC, as described in Materials and methods. Arrows indicate the elution positions of standards.
Figure 4. Sequence of CEFT-1. (A) The predicted nucleotide and deduced amino acid sequence of the CEFT-1 cDNA are shown. The adenine residue of the putative initiation codon is assigned as 1, while the amino acids encoded by the cDNA are depicted by single letter code. (B) Hydropathy plot (41) of the 451-amino acid polypeptide predicted by the CEFT-1 DNA sequence.
Figure 5. (A) Comparison of the protein sequences of CEFT-1 with vertebrate [alpha]1,3FTs. Comparisons are shown for human FTIII, bovine FT (bFT), chicken FT-1 (cFT-1) and the C.elegans CEFT-1. Amino acid identities are indicated by dark shading and boldface letters, while identities between CEFT-1 and 2 other enzymes are indicated by shading alone. The boxed sequences represent the predicted transmembrane domain of the CEFT-1 protein. (B) The 5[prime] exon/intron sequences of the isolated CEFT-1. A portion of the exon 1 sequence is shown underlined and spans nucleotides 61 through 89. The exon 2 sequence shown is also underlined and begins at nucleotide 424. The partial sequence of intron 1 found in the isolated cDNA spans from nucleotides 90 through 167. The boxed area and the arrow indicate recognition and cleavage site for BspE1.
Figure 6. Southern and Northern blot analysis of C.elegans genomic DNA and total RNA. (A) High molecular weight genomic DNA was isolated from C.elegans adults, subjected to restriction enzyme digestion, fractionated on agarose gels, and transferred to nylon membranes. Following prehybridization and hybridization with 32P-labeled CEFT-1 cDNA, blots were washed with high stringency as described in Materials and methods and subjected to autoradiography. Restriction enzymes are indicated. (B) Total RNA from C.elegans adults (12, 25, or 60 µg) was separated on an agarose gel containing formaldehyde, and subsequently transferred to nylon membranes. Hybridization was carried out using isolated CEFT-1 cDNA, as described in Materials and methods, and the blots were autoradiographed. Size markers in kbp are indicated.
Source of cDNA
Product formationa
c.p.m.
pmol/mg·h
Mock-transfected
40
<7.0
C.elegans CEFT-1
(+ partial intron 1)200
33.3
C.elegans CEFT-1
(- partial intron 1)818
140
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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