Glycobiology, 2002, Vol. 12, No. 5 299-306
© 2002 Oxford University Press
The presence of Lewis a epitopes in Arabidopsis thaliana glycoconjugates depends on an active 
4-fucosyltransferase gene
2 Equipe de Glycobiologie et Biotechnologie (EA 3176), Institut des Sciences de la Vie et de la Santé, Université de Limoges, Faculté des Sciences, 123, Avenue Albert Thomas, 87060 Limoges, France; 3 Laboratoire de Physiologie et de Biochimie Végétales URA CNRS 571, Université de Poitiers, 25 rue du Faubourg St Cyprien, 86000, Poitiers, France; and 4 Laboratoire des transports intracellulaires, CNRS UMR 6037, IFRMP 23 Université de Rouen, UFR des Sciences, 76821 Mont St Aignan, Cedex, France
Received on October 30, 2001; accepted on February 13, 2002.
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The presence of an
4-fucosyltransferase in plants was first deduced from the characterization of Lewis-a glycoepitopes in some N-glycans. The first plant gene encoding an 4-fucosyltransferase was recently cloned in Beta vulgaris. In the present paper we provide evidence for the presence of an 4-fucosyltransferase in A. thaliana by measurement of this glycosyltransferase activity from a purified microsomal preparation and by immunolocalization of Lea epitopes on glycans N-linked to glycoproteins located to the Golgi apparatus and on the cell surface. The corresponding gene AtFT4 (AY026941) was characterized. A unique copy of this gene was found in A. thaliana genome, and a single AtFT4 transcript was revealed in leaves, in roots, and at a lower extent in flowers. The coding sequence of AtFT4 gene is interrupted by two introns spanning 465 bp and 84 bp, respectively. The putative 393-amino-acid protein (44 kDa, pI: 6.59) contains an N-terminal hydrophobic region and one potential N-glycosylation site, but AtFT4 has poor homology (less than 30%) to the other 3/4-fucosyltransferases except for motif II. When expressed in COS 7 cells the protein is able to transfer Fuc from GDP-Fuc to a type 1 acceptor substrate, but this transferase activity is detected only in the culture medium of transfected cells
Key words: 3/4 fucosyltransferase/Arabidopsis thaliana/Lewis a/Silene alba
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Introduction |
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Fucosyltransferases catalyse the transfer of fucose from GDP-fucose to oligosaccharide acceptors linked to proteins, lipids, and sugars (Costache et al., 1997
). The 1,2, 1,3, and 1,4 linkages of fucosyl residues are present in plants. The Lewis a (Lea) determinant is generated by the 1,4 linkage (Fitchette-Laine et al., 1997; Lhernould et al., 1997; Melo et al., 1997), whereas 1,3 linkage contributes to form the core N-glycans. 3-Fucosyltransferase transfers L-fucose on the nonreduced GlcNAc residue of the chitobiose, whereas the reduced GlcNAc of lactosamine complex N-glycan is the substrate for 4-fucosyltransferase. Fucosylated N-glycans are widely observed in mammalian (Lowe et al., 1990), insect (Staudacher et al., 1991, 1992; Staudacher and Marz, 1998) and plant glycoproteins (Roberts and Lord, 1981; Staudacher et al., 1995; Altmann, 1998; Leiter et al., 1999; Lerouge et al., 1998; Fitchette-Laine et al., 1997; Melo et al., 1997; Rayon et al., 1999; Zeleny et al., 1999; Van Die et al., 1999). Core N-glycans fucosylated in 1,6 and 1,3 are found in mammalian and in plant cells, respectively (Staudacher et al., 1999), and they are both present in insect cells (Staudacher and Marz, 1998). The maturation of plant complex N-glycans is governed by Golgi-anchored glycosyltransferases, but the detailed process of this maturation is still a matter of discussion (Kimura et al., 1987; Lerouge et al., 1998). Nevertheless, it is generally accepted that core-fucosylation occurs during the early steps of complex N-glycan maturation whereas Lewis-fucosylation is a late event (Fitchette-Laine et al., 1997; Fitchette et al., 1999).
All known animal 3/4-fucosyltransferases are type II membrane proteins composed of cytoplasmic, transmembrane, hypervariable, and catalytic domains. The catalytic domain contains two highly conserved peptide sequences named motifs I and II, which seem to be involved in GDP-Fuc recognition (Breton et al., 1998; Oriol et al., 1999). Leiter et al. (1999) reported that the core 3-fucosyltransferase of Vigna radiata and the mammalian 3/4-fucosyltransferases have four common regions. Among them, two regions correspond to the characterized motifs I and II. The third sequence is named region A (153-LPQPSGTASILRSMESA) and is located 13 amino acids before motif I. The last common sequence is named region C (226-ISNCGARNFRLQALEALEKSNIKIDSYGG) and is located between motifs I and II.
Fucosylation in (1,4) linkage is well demonstrated in primates as confirmed by the presence of Lea and by the characterization of human and chimpanzee genes encoding 4-fucosyltransferases (Oriol et al., 1999). However, in the plant kingdom, the detection of Lea epitopes was indirect proof of 4-fucosyltransferases activity in plant tissues (Fitchette-Laine et al., 1997; Wilson et al., 2001b). The present article provides demonstration of the occurrence of 4-fucosylation in Arabidopsis thaliana through Lea immunodetection and -4FucT activity measurement. Cloning and expression of AtFT4 in COS-7 cells show that the gene encodes an enzyme able to transfer fucose in 1,4 linkage to type 1 acceptor substrates. While this work was under revision, Bakker et al. (2001) described a gene of Beta vulgaris coding 4-FucT.
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Lea detection and immunolocalization in A. thaliana cells
Plant anti-Lea antibodies reacted with N-glycoproteins in crude protein extracts obtained from A. thaliana plants and suspension-cultured cells (Figure 1). The immunoreaction was specific because the labeling disappeared after absorption of plant anti-Lea antibodies by Lea epitopes present in human saliva (Figure 1). Immunodetection analysis revealed that the pattern of Lea glycoproteins in cultured cells differs from that of plants. The differential expression between cell suspension and plant tissues suggests that glycoproteins carrying Lea epitopes are probably different and could be implicated in several metabolic pathways.
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Electron microscopy of A. thaliana cells treated for immunogold staining with plant anti-Lea antibodies showed a clear distribution of Lea determinant in plasma membrane (Figure 2A) and in Golgi-derivated vesicles (Figure 2B). This result is in good agreement with the previous observations reported for tomato, tobacco, onion, and maize (Fitchette et al., 1999
). A preincubation of anti-Lea antibodies with Lea-positive human saliva induced the loss of the immunogold labeling (data not shown). Therefore, Lea-containing glycoproteins are detectable in A. thaliana, and our data support their location to the plasma membrane and the Golgi apparatus.
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(1,4)-Fucosyltransferase activity in A. thalianaEnzyme activities were determined using crude protein extracts from cells and microsomes using the acceptor substrates type 1, H-type 1, and type 2. Type 1 acceptor substrate (Galß1,3GlcNAc) can be fucosylated either in
1,2 on Gal or in 1,4 on GlcNAc. The trisaccharide H-type 1 (Fuc1,2Galß1,3GlcNAc) is an appropriate acceptor substrate to analyze specifically the formation of 1,4 fucose linkage on GlcNAc. No fucosyltransferase activity was detected in A. thaliana cell crude protein extract using type 1 and H-type 1 (Table I). In contrast, a protein extract obtained from S. alba cells showed a significant enzyme activity that reached 32 pmol min–1 mg–1 protein. Because the activity was almost identical with both acceptors, we concluded that only 1,4-fucosyltransferase activity was measured in our experimental conditions. Because all known fucosyltransferases are Golgi-anchored proteins, the enzyme activity was determined from microsomal fractions. 1,4-Fucosyltransferase activity was detected in microsomes purified from A. thaliana (50 for type 1 and 48 pmol min–1 mg–1 protein for H-type 1). As expected, enzyme activity in S. alba microsomes (260 for type 1 and 248 pmol min–1 mg–1 protein for H-type 1) was higher than that measured in cell crude extract. Moreover, these results revealed that 1,4-fucosyltransferase activity in S. alba was about five times higher than in A. thaliana. Neither crude nor microsome S. alba or A. thaliana extracts exhibited fucosyltransferase activity with the type 2 acceptor substrate (Galß1,4GlcNAc) (Table I).
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These results clearly demonstrate that A. thaliana cells contain an active
4-fucosyltransferase even if its expression is much weaker than in S. alba cells. To confirm that Fuc is 1,4 linked to GlcNAc of type 1 acceptor substrate, a composition and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) analysis were performed. MALDI-TOF mass spectrum of the pseudomolecular ion (M+H)K+ was detected at m/z 664, which was consistent with the predicted value calculated from the Lea structural motif (calculated average mass m/z + K+ 664.7). The linkage position of a Fuc to the type I acceptor substrate was then unequivocally elucidated from the diagnostic ions of the EI mass spectra of the partially methylated, partially acetylated methyl glycosides obtained from the transfer product. The mass spectrum analysis, confirmed the formation of 6-methyl-3,4-acetyl-GlcNAcMe (which may be recognized from the main ion fragments m/z 115, 170, and 256), per-O-methyl-Fuc and per-O-methyl-Gal, indicating that Fuc was linked to the GlcNAc residue at the O-4 position.
cDNA cloning
TBlastN search in DDBJ A. thaliana genome database (available online at http://arabidopsis.org) of fucosyltransferase motif II HYKFSLAFENSNE-EDYVTEKFF-QSLVAGTVPVV revealed three putative genes corresponding to the sequences FucTA (accession no. AP000419), FucTB (accession no. AC011807) and FucTC (accession no. AC021665). As described by Wilson et al. (2001a), FucTA and FucTB encode core 1,3 fucosyltranferases. However, no information was available on the putative function of FucTC. The complete sequence of FucTC has five putative start codons. We have chosen to amplify an open reading frame (ORF) of 1179 bp, named AtFT4, including the third ATG of FucTC as a start codon, because it is the only one conventionally positioned (Kozak, 1981). The protein encoded by AtFT4 has a hydrophobic amino terminal domain of 16 residues (M1–P16) probably playing the role of a transmembrane domain but without a cytosolic tail (Figure 3). Oligonucleotides (S1; S2; and the nested ones, S3, S4) including start and stop codons were designed and used as primers for polymerase chain reaction (PCR) amplification of the AtFT4 cDNA from 10-day-old A. thaliana leaves. One fragment of approximately 1200 bp was obtained. After DNA sequencing, it was found to be strictly identical to the predicted ORF of the genomic DNA. PCR amplification of the same sequence on genomic DNA gave a longer fragment (1728 bp) than the cDNA (1179 bp), suggesting the presence of intronic sequences in the gene. Three exons (E1, E2, E3) and two introns (I1, I2) spanning 465 bp and 84 bp, respectively, were identified (Figure 4).
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The ORF of AtFT4 gene (accession no. AY026941) encodes a putative 393-amino-acid protein with a calculated molecular mass of 44 kDa and pI of 6.59 (http://www.expasy.ch/tools/pi_tool.html) (Figure 3). Only one potential site of N-glycosylation is found at position N-77 in the A. thaliana sequence and one in B. vulgaris (Bv-FT4, position N-83) compared with the two sites in Vigna radiata
1,3 fucosyltransferase (N-346, N-390) and the three sites in FucTA and FucTB core 1,3fucosyltransferases.
Southern blot and northern blot analyses
A mono-exonic probe (1179 bp) was used in hybridization studies with genomic DNA of two ecotypes of A. thaliana (cv landsberg and cv columbia) digested with EcoRI. A single band of about 3 kb was detected for each A. thaliana digested DNA suggesting the presence of only one 4-fucosyltransferase gene in the A. thaliana genome (Figure 5A). Another digest of genomic DNA with EcoRI/BamHI and an exhaustive search in DDBJ database confirmed that A. thaliana genome contains a single copy of AtFT4 (data not shown).
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Northern blot analysis of mRNA isolated from various A. thaliana cv columbia tissues revealed the presence of a transcript of about 1.2 kb (Figure 5B). All analyzed tissues contained AtFT4 transcript. Densitometric analysis of the bands suggested that leaves have higher levels of AtFT4 transcripts (100%) compared with roots (60%) and flowers (40%).
Expression of AtFT4 in mammalian COS-7 cells
The AtFT4 cDNA was subcloned into the mammalian expression vector pTARGET and transiently transfected into COS-7 cells to determine the activity of the enzyme encoded by AtFT4 in vitro. The fucosyltransferase activities were measured in the cellular homogenates and in the culture medium of COS-7 cells using Galß1,4GlcNAc-Biotin (type 2), Galß1,3GlcNAc-Biotin (type 1), and Fuc1,2Galß1,3GlcNAc-Biotin (H-type 1) as acceptor substrates.
No fucosyltransferase activity was detected in the protein extract of COS-7 cells transfected with pTARGET/AtFT4 whatever the acceptor substrate. Nevertheless, culture medium proteins were able to transfer Fuc on type 1 (0.2 pmol min–1 mg–1 protein) and on H-type 1 (0.2 pmol min–1 mg–1 protein) but not on type 2 acceptors. Our results demonstrate that the A. thaliana protein encoded by AtFT4 cDNA is an enzyme secreted into the culture medium of transfected COS-7 cells and is able to transfer Fuc in (1,4)-linkage. Kinetic studies (Table II) showed that Fuc transfer from GDP-Fuc to Type 1 acceptor progressively increased with the incubation time. Varying the protein concentration within the reaction mixture proportionally changed the fucosyltransferase activity. These results confirm the enzymatic properties of the protein encoded by AtFT4.
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We report the presence of the Lea determinant in A. thaliana cells. Lea epitope was localized on the surface of A. thaliana plasma membrane. The
4-fucosyltransferase gene (AtFT4) was cloned and expressed in COS-7 cells. AtFT4 ORF (AY026941) encodes a putative 393-amino-acid protein (AtFT4). AtFT4 protein is approximately 100 amino acids shorter than the other known plant FucT but has a similar length as animal (1,3/4)-FucT (Staudacher et al., 1999). AtFT4 NH2-terminal is eight amino acids shorter than FucTC (Wilson et al., 2001a). Hydrophobicity analysis (Kyte Doolittle, Hopp Wood, Eisenberg) of AtFT4 reveals no cytoplasmic tail but an NH2-terminal hydrophobic domain that may correspond to the transmembrane domain of the protein. The truncated form of FucTC, lacking the cytoplasmic tail and the transmembrane domain (first 30 N-terminal amino acids) was expressed in Pichia pastoris, but no activity was detected (Wilson et al., 2001a). The heterologous system they used and/or the low level of activity they measured for the product of this gene could explain their result. In contrast, AtFT4 is shorter than FucTC, but longer than the truncated form described by Wilson et al. (2001a) and encodes an enzyme that uses a type 1 acceptor substrate. In our system, the fucosyltransferase activity was found in the culture medium of the COS-7-transfected cells. A computer analysis suggested the occurrence of a signal peptide cleavage site between T28 and S29.
Finer analysis of the peptide sequence of AtFT4 showed that motif II defined in mammalian lactosamine-(3/4)-fucosyltransferases is conserved between animal and plant (55% identity) (Figure 6). This motif seems to be involved in GDP-Fuc binding. We recently characterized the acceptor motif of vertebrate fucosyltransferases (Dupuy et al., 1999). One amino acid (W) is involved in enzyme specificity (-HHWD- for lactosamine-1,4-fucosyltransferase, and –HHRD- for lactosamine-1,3-fucosyltransferase). AtFT4 contains a sequence 57-VLVAYKKWD-67, which may correspond to the acceptor-binding motif (Figure 3). This motif is also found in the B. vulgaris 4-FucT protein (56-LLGAFRKWD-64), but is absent in FucTA, FucTB, and V. radiata FucT c3, which are core -(1,3)-fucosyltransferases. However, two conserved cysteins (C-377 and C-380) corresponding to the residues involved in disulfide bonds of the lactosamine-fucosyltransferases (Holmes et al., 2000) were also found in AtFT4.
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Computer analysis of AtFT4 homologs showed eight putative proteins: tomato developing/immature green fruit EST (accession no. BF051851), Lotus japonicum EST (accession no. AV418585), barley seedling root EST (accession no. BF255044), Gossypium hirsutum (accession no. AW186741), Solanum tuberosum (accession no. AW906413), Glycina max (accession no. BG726765), B. vulgaris (accession no. AJ315840), and Zea mays (accession no. BG 874074). All these sequences shared more than 85% identity with the peptide sequence of AtFT4. These data suggest that peptide sequences of plant
4-FucT are highly conserved and that they differ significantly from the primate enzymes (less than 10% identity) even if a similar activity is maintained due to some conserved motifs. Questions arise about the origin of these motifs. Are they the products of a common ancestral duplication with a divergent evolution in animal and plant kingdoms? Or are they the result of independent evolution of at least two genes (one for plants and one for animals) with a selective pressure around short sequences defined now as markers for this activity? Plant 4-FucT activity is broadly distributed in the plant kingdom as shown by EST results but shows a differential expression during plant differentiation and development. The role played by 4-fucosylation in these processes remains to be elucidated. Further investigations are needed to compare plant fucosylation with the results collected for this activity in mammals. |
Materials and methods |
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Plant cultivar
A. thaliana cv columbia or cv landsberg were grown in a greenhouse and harvested at different stages corresponding to rosette formation or flower development. Cell cultures of A. thaliana cv columbia (plantlets calli were a gift from B. Lescure, CNRS-INRA Castenet-Tolosan) were grown in a climate chamber under continuous light (80 µE m–2 s–1) and rotation (120 rpm) in a vertical flask agitator. Culture medium (Camborg B5) was supplemented with 20 g L–1 sucrose.
Molecular standard methods
Plant nucleic acids of shoots of 10-day-old plants were prepared with the respective DNeasy and RNeasy kits from Qiagen GmbH (Hilden, Germany). Genomic and cDNA sequences of AtFT4 were amplified by nested PCR with two sets of primers: S1, 5'-AGGAATCAATCACACCATGCC-3' and S2, 5'-GCAATGGCCGCTCTACTAATG-3'; and S3, 5'-GCAATGGCCGCTCTACTAATG-3' and S4, 5'-CATCAAACTCCGGCGTTCTTCCC-3'). Before sequencing, PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Transfections of COS-7 cells were carried out using the pTARGET expression vector (Promega).
Southern blot and northern blot analysis
Arabidopsis genomic DNA was digested with EcoRI or EcoRI/BamHI and fractionated by electrophoresis through 0.8% (w/v) agarose gel. After depurination (15 min) with 0.25 N HCl and denaturation (30 min) with 0.4 N NaOH, DNA was transferred on Hybond-N+ membranes (Amersham, Arlington Heights, IL).
RNA from Arabidopsis tissues was isolated using the Qiagen Plant Mini Kit (Qiagen). For northern blot analysis, RNA was size-fractionated on formaldehyde agarose gels and transferred to Hybond-N+ membranes (Amersham).
Hybridization was carried out with a 1179-bp monoexonic probe generated by PCR amplification on cDNA. Twenty-five nanograms of probe were labeled with PCR DIG Probe Synthesis Kit (Boehringer-Mannheim, Germany). Hybridization was carried out overnight at 42°C in DIG Easy Hyb (Boehringer-Mannheim). Blots were washed three times for 15 min each at 65°C with 2x SSC/0.1% sodium dodecyl sulfate (SDS), 1x SSC/0.1% SDS, and 0.2x SSC/ 0.1% SDS, then analyzed after exposure to X-ray films (Kodak, Kyoto, Japan) for 1 h at –80°C.
DNA sequencing
Sequencing was performed using T7 promoter and pUCM13 reverse primers for DNA cloned into pGEM-T Easy vector or directly with primers used for PCR amplification. A dye labeling chemistry (kit PRISM Ready Reaction Ampli Taq FS) and the ABIPrism 310 Genetic Analyzer (Perkin Elmer, Norwalk, CT) were used.
Transient expression of Arabidopsis cDNA
The full-length AtFT4 cDNA (1,240 bp) was inserted into the pTARGET (pTARGET/AtFT4) to transiently transfect COS-7 cells. SuperFect transfection reagent (Qiagen) was used according to the protocol described by the manufacturer. After 48 h, COS-7 cells were harvested and washed with phosphate buffered saline (PBS), and proteins were subsequently extracted in lysis buffer (1% [v/v] Triton X100, 10 mM sodium cacodylate [pH 6], 20% [v/v] glycerol, 1 mM dithiothreitol) for 2 h at 48°C. The suspension was then centrifuged at 12,000 x g for 10 min at 4°C, and the supernatant was used for assays. Media of transfected and not transfected COS-7 cells were concentrated before use for enzymatic assays.
3/4-Fucosyltransferase assay
Fucosyltransferase assays were conducted in 60 ml containing 25 mM sodium cacodylate (pH 6.5), 5 mM ATP, 20 mM MnCl2, 10 mM -L-fucose, 3 µM GDP-[14C]-fucose (310 mCi/mmol; Amersham), and 50 µg of proteins (from crude extract or supernatant of transfected COS-7 cells). The mixture was incubated 1 h or 3 h at 37°C. Acceptor substrates (type 1, type 2, and H-type 1 from Syntesome, Munich) were used at the concentration of 0.1 mM. The reaction was stopped by addition of 3 ml cold water. The reaction mixture was then applied to a conditioned Sep-Pak C18 reverse chromatography cartridge (Waters Millipore, Bedford, MA). Unreacted GDP-[14C]-fucose was washed off with 15 ml of water. The radiolabeled reaction product was eluted with 2x 5 ml ethanol, collected directly into scintillation vials, and counted with two volumes of biodegradable counting scintillant (Amersham) in a liquid scintillation beta counter (Liquid scintillation analyzer, Tri-Carb-2100TR, Packard).
Immunocytochemical procedure
The antibodies used were raised against the plant Lea glycoepitope (Fitchette-Laine et al., 1997).
A. thaliana cells were fixed for 15–30 min in a mixture of 1.5% (w/v) paraformaldehyde and 0.5% glutaraldehyde in 50 mM phosphate buffer, pH 7.2 (Fleurat-Lessard et al., 1995). Abundant washing in the same buffer was followed by 4 min postfixation in 1% (v/v) OSO4, dehydratation in ethanol series, and overnight embedding in London Resin White. Polymerization occurred in gelatin capsules at 60°C for 24 h.
The immunogold reaction on thin sections, carefully spread with toluene vapor on parlodion-coated gold grids, was performed at 20°C, as previously described (Bouché-Pillon et al., 1994a,b). The procedure accommodated a compromise between the preservation of structure and antigenicity. Solution was filtered (0.1 µm pores, Millipore MFVCWP). The sections, hydrated in deionized water, were then in the dark etched by 0.56 M NaIO4 and 0.1 N HCl, and washed for 15 min with PBS, 0.1%(v/v) Triton X-100, and 0.2% (v/v) Gly at pH 7.2. Nonspecific sites were saturated for 45 min by goat serum in PBS, 0.2% (v/v) Triton X-100, 0.2% (v/v) Tween 20, and 0.1% (w/v) bovine serum albumin (BSA); sections were incubated overnight with the Lea antibody at a 1:50 dilution. After washing in PBS, sections were placed for 40 min on Tris-buffered saline (pH 8.2) 0.2% (v/v) Tween, 0.2% (v/v) Triton X-100, 1% (w/v) BSA, and goat serum before the 3-h application of a 15-nm gold particle labeled goat anti-mouse IgG at a 1:40 dilution. The sections, washed in Tris-buffered saline and deionized water, were contrasted in uranyl acetate at saturation in water and in lead citrate. Controls were as for the semithin sections. Specimens were observed with an electron microscope (100C, JEOL) operated at 80 KV.
Protein extraction and western blot experiments
Cells were harvested by filtration on Whatman 41 paper with a Büchner funnel and disrupted with liquid nitrogen.
The protein fraction was extracted into cacodylate buffer (200 mM sodium cacodylate, pH 7.0, containing 1% (w/v) Triton X-100; Sigma). The homogenate was centrifuged at 14,000 x g for 30 min. The supernatant constitutes the crude protein extract. For microsome preparation, disrupted cells were homogenized on ice in 200 mM HEPES-KOH buffer (pH 7) containing 1 mM dithiothreitol and 0.4 M sucrose (Misawa et al., 1996). The homogenate was filtered through cloth nylon and centrifuged at 5000 x g for 10 min at 4°C. The supernatant was then centrifuged again at 100,000 x g for 1 h at 4°C (Beckman L8-80). The pellet was then resuspended in cacodylate buffer.
Protein extracts were obtained by homogenizing plant material in a solution containing 0.7 M sucrose, 0.5 M Tris, 30 mM HCl, and 2% (v/v) ß-mercaptoethanol. After incubation on ice for 30 min, the homogenate was centrifuged for 5 min at 5000 x g. The supernatant was mixed vigorously with one volume of saturated phenol, left on ice for at least 30 min, and centrifuged at 10,000 x g for 30 min. The upper phenolic phase was precipitated overnight at 4°C by the addition of five volumes of methanol containing 0.1 M ammonium acetate. The preparation was then centrifuged for 30 min at 10,000 x g. The pellet was washed once with 0.1 M ammonium acetate in methanol and twice with acetone before being resuspended in sample buffer (62.5 mM Tris–HCl, pH 6.8, containing 10 mM dithiothreitol, 10% [v/v] glycerol).
Protein amount was determined by the Bradford method using BSA as a standard (Bradford, 1976).
The crude extracts were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) using a 12% acrylamide Tris-glycine gel (Laemmli, 1970). Proteins were visualized by Coomassie blue staining and transferred onto nitrocellulose membrane (Schleicher & Schuell, Germany). Membrane coating was performed overnight at 4°C with Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) BSA (TBS-T-BSA). After three washings in TBS-T, the membrane was incubated for 1 h at 20°C with a mouse anti-Lea antibody in TBS-T-BSA buffer at 1:1000 dilutions. Rabbit anti-mouse Ig coupled to horseradish peroxidase (Amersham, England), was used at 1:1000 dilution. The western blots were developed according to the ECL detection kit (Amersham).
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAbbreviationsReferences |
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BSA, bovine serum albumin; Lea, Lewis a; MALDI-TOF MS, matrix-assisted laser desorption time-of-flight mass spectrometry; ORF, open reading frame; PBS, phosphate buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, sodium sodium citrate; TBS-T-BSA, Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) BSA.
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1 To whom correspondence should be addressed
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References |
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