Department of Pharmacology and Molecular Sciences and 3Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
Received on August 14, 2000; revised on November 3, 2000; accepted on November 3, 2000.
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Abstract |
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Key words: FACE/fucose/glycosyltransferase/iminosugar/Schistosome/trematode
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Introduction |
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As the parasites mature, they follow a specific path within the mammalian host to complete their life cycle. Fucosylation may regulate their trafficking during development, as is the case for certain mammalian saccharide structures on blood cells (Maly et al., 1996; Le Marer et al., 1997
). Fucosylated glycoconjugates are also important in the host immune response to the parasite. Some of the schistosome eggs are trapped in the host tissues, and the immune response to the egg fucosecontaining epitopes results in the formation of granulomas. Among the most potent inducers of granuloma formation are fucose-containing glycoconjugates unique to schistosomes and recognized by mAb 128/C3 (Weiss and Strand, 1985
; Weiss et al., 1986
, 1987; Levery et al., 1992
).
Schistosomal fucosylated structures contain 1,3 fucose linkages and other types, including Fuc
1,2Fuc, Fuc
1,6GlcNAc, and an unusual internal linkage, Fuc
1,4GlcNAc (Levery et al., 1992
). S. mansoni fucosyltransferase activities have been investigated in extracts of adult worms with the use of a small number of acceptor substrates (LNnT, LNT, NeuAc
2,3Galß1,4GlcNAc, and NeuAc
2,6Galß1,4GlcNAc; see Table I for acceptor substrate abbreviations) (DeBose-Boyd et al., 1996
). These extracts contained a fucosyltransferase that preferentially fucosylated the acceptor LNnT, forming the Lewis x (LeX) determinant, and also showed an activity capable of synthesizing sialyl Lewis x (sLeX). The fucosyltransferase activity in these crude extracts had an apparent KM of 300 µM for GDP-Fuc and a Vmax of 0.23 nmol/mg/h. Fucosyltransferase activities have also been studied in the avian species of Schistosoma, Trichobilharzia ocellata, where
3- and
2-fucosyltransferases were the main activities identified. One of the products formed by these T. ocellata transferases was determined to be Fuc
1,2Fuc
1,3GalNAcß1,4GlcNAc (Hokke et al., 1998
). A fucosyltransferase encoded by S. mansoni with a high percent of sequence identity and enzymatic activity comparable to human and mouse fucosyltransferase VII has been cloned (Marques et al., 1998
). More recently, another S. mansoni cDNA sequence with similarities to mammalian fucosyltransferases was isolated, but its enzymatic activity has not yet been determined (Trottein et al., 2000
).
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Results |
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The schistosome core I structure, GalNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc, was based on the carbohydrate structure of the schistosome neutral egg glycolipids identified by Khoo et al. (1997). It was predicted that the oligosaccharide backbone of that structure would be the natural substrate for the schistosomal fucosyltransferases. This oligosaccharide was biosynthetically made using ß1,4-galactosyltransferase, which mediates the transfer of GalNAc to the nonreducing end of chitotriose to yield schistosome core I. The addition of GalNAc to chitotriose was determined using fluorophore-assisted carbohydrate electrophoreses (FACE). Previous enzymatic syntheses using this procedure have resulted exclusively in ß-linked terminal GalNAc (Palcic and Hindsgaul, 1991
; Do et al., 1995
).
FACE fucosyltransferase assay
The use of FACE to separate the substrates and products of the fucosyltransferase assay allowed quantitative analyses of reaction products. Furthermore, multiple radiolabeled products in a single reaction could be detected and quantified. The FACE fucosyltransferase assay was first optimized using commercial enzymes and compared to standard methods (data not shown). To quantify radioactivity in the products we used a radioactive scale that was exposed simultaneously with the phosphorimager plate. The procedure demonstrated a linear correlation between pixel density and d.p.m. The lowest concentration of radioactivity, which yielded pixel density above background, was 2.5 d.p.m. We set the pixel density of the 5 d.p.m. standard as the limit of detection for all subsequent experiments.
A limitation of the current assay is that product characterization is not definitive. However, transfer of radiolabeled fucose to a well-defined acceptor, combined with product electrophoretic mobility compared to known standards, allowed tentative assignments to be made in many cases.
When schistosome extracts were incubated with GDP-[14C]-fucose and the ANTS-labeled acceptor oligosaccharide, LNnT, a linear correlation was observed between the amount of product formed and the amount of protein extract from schistosome that was used in the reaction mixture. As expected, the quantity of product formed was found to increase with time (Figure 1A and B). The multiple products formed using this acceptor are more fully described in Fucosyltransferase and fucosidase activities are regulated during S.mansoni development.
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An example of the fucosyltransferase data is shown in Figure 2, and a summary of the activities of each acceptor in extracts of cercariae, adult worms, and eggs is shown in Table II. For all life stages tested, the oligosaccharide LNnT was the best acceptor. Surprisingly, regardless of the acceptor, the fucosyltransferase-specific activities of the egg extracts were higher than that of the adult worms or cercarial extracts, with the activity for any specific acceptor varying up to 100-fold from one life stage to another. For example, the use of SLNnT as substrate yielded a specific activity of 52.7 pmol/mg/h for the egg extract, 13-fold the activity observed with the use of the LNFIII for this life stage, while the specific activity for the SLNnT was only 0.5 pmol/mg/h in an extract of adult worms and is equivalent to the activity for LNFIII in the same extract. In contrast, fucosyltransferase activity for some other acceptors varied by only two- to fourfold from one life stage to another. For example, the specific activity for the acceptor LNFIII was 4 pmol/mg/h for the egg extract, 1.8 pmol/mg/h for the cercarial extract, and 0.5 pmol/mg/h for the adult worm extract. Some acceptor oligosaccharides, such as 3'F-Lac, only showed activity in egg. The differences of fucosyltransferase activities in the life stages indicates the presence of more than one enzyme in the extracts and also suggests that specific enzymes and fucosylated products may exert their main functions during particular life stages.
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Chitotriose (GlcNAcß1,4GlcNAcß1,4GlcNAc) and chitotetraose (GlcNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc) were also acceptors, each producing a single characteristic radioactive band. The smaller saccharide chitobiose (GlcNAcß1,4GlcNAc) was apparently not used as an acceptor substrate by the Schistosoma. The saccharide blood group A (GalNAc1,3(Fuc
1,2)Gal) was not a substrate. Schistosome core I (GalNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc) was the third-best acceptor identified for the schistosome egg extracts (Table II), and it formed more than one radioactive product, possibly including the putative epitope of the mAb 128C3/3, although more experiments are required to test this hypothesis. No products were detected when schistosome core I was tested using adult or cercaria extracts.
For some substrates, especially those that are both sialylated and fucosylated, such as sLex, sLea, and SF-Lac, there were several visible fluorescent bands that had more rapid electrophoretic mobility than that of the starting oligosaccharide and were absent from the control reactions (Figure 2; fluorescence). These bands may be degradation products catalyzed by glycosidases present in the schistosome extract. However, neither these sugars nor their degradation products yielded radioactive fucosylated products. Several other fluorescent bands with slower migration were also visible in the reactions with several of the substrates. In most cases these do not appear to be fucosylation products, since they do not appear as corresponding fucosylated bands on the autoradiograph; therefore, they may be products of other reactions. For example, desialylation, reduces the total negative charge of an oligosaccharide reducing its migration.
Because fucosidase activities in the schistosome extracts may interfere with the interpretation of the fucosyltransferase data, 2- and
3-fucosidase activities were determined (Figure 4). Fucosidase activity was determined by measuring the formation rate of lactose from the oligosaccharides 3'F-Lac (Galß1,4(Fuc
1,3)Glc) (Figure 4C) and 2'F-Lac (Fuc
1,2Gal-ß1,4Glc) (Figure 4B) in the presence of schistosomal extracts. ANTS-labeled lactose migrates differently than do the ANTS-labeled fucosylated oligosaccharides (Figure 4A). Both
2- (Figure 4B)
3-fucosidase (Figure 4C) activities were detected. The results are summarized in Table III. In these experiments the egg extracts showed higher
2- and
3-fucosidase-specific activities than the other life stages did. This result indicates that the higher fucosyltransferase activity detected in egg extract is not due to lower fucosidase activity in this extract. It is noteworthy that
3-fucosidase activity was 2.5- and 10-fold greater than
2-fucosidase activity in the cercarial and adult worm extracts, respectively. In contrast, the
3-fucosidase activity in the egg extract was only one third of the
2-fucosidase activity. These high fucosidase activities at substrate concentrations of 0.5 mM may interfere with the determination of the fucosyltransferase activity by degrading the products, resulting in underestimation of the fucosyltransferase activities. However, the fucosylated substrates, LNFII, LNFIII, and LNDI did not display degradation products, nor did the sialylated substrates LSTa or LSTc.
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Discussion |
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In general, the schistosome fucosyltransferases prefer type-2 uncharged sugars, such as LNnT, as acceptors and may preferentially form 1,3 linkages (Figure 2; LNnT and Figure 3). The type-2 sialylated oligosaccharides, SLNnT and LSTc, also showed significant activity as acceptor substrates when incubated with egg extracts. Fucosylation of these oligosaccharides may form the sLex structure. The formation of sLex by schistosome fucosyltransferases extracts (DeBose-Boyd et al., 1996
) and a fucosyltransferase-type VII encoded by the S. mansoni able to express the sLex epitope in recombinant cells (Marques et al., 1998
) have been reported previously. In addition, L-selectin, a high-affinity lectin for sLex, has been found bound to egg miracidia (El Ridi et al., 1996
).
Schistosomal extracts could also fucosylate LNFIII, forming a product that comigrated with LNDI, which is a hexasaccharide containing two fucoses. The LNFIII-fucosylated product may contain Fuc1,2Fuc in that the LNFIII oligosaccharide is similar to the acceptor substrate used by Hokke and collaborators (Hokke et al., 1998
) to identify the Fuc
1,2Fuc fucosyltransferase. An alternative possibility is that the fucosylated structure formed is Fuc
1,2Gal, but this structure seems less likely because the other acceptor substrates that terminate in Galß1,4GlcNAc did not serve as substrates.
3'F-Lac, which terminates in Galß1,4Glc, was used as an acceptor by the schistosomal fucosyltransferases, and the SF-Lac, 2'F-Lac and Lex were not. These results suggest that there is a fucosyltransferase that can form Fuc1,2Galß1,4(Fuc
1,3)Glc but may not form Fuc
1,2Galß1,4(Fuc
1,3)GlcNAc. It is interesting that a lectin has been identified in the intermediate host snail that binds to Fuc
1,2Galß1,4Glc, and this saccharide is known to be highly expressed in the egg miracidia. This interaction between the snail lectin and the miracidial sugar Fuc
1,2Galß1,4Glc may play a role in mediating the infection of the intermediate host (Mansour et al., 1995
; Mansour, 1996
). Alternatively, the product formed using 3'F-Lac as acceptor may contain a Fuc
1,2Fuc linkage.
Chitotriose, chitotetraose, and schistosome core I were also substrates for the schistosomal fucosyltransferases. These oligosaccharides resemble the repetitive units of the putative epitope of mAb 128C3/3. Chitotetraose and schistosome core I differ only in their nonreducing terminal saccharide (GlcNAc vs. GalNac). This difference was apparently significant, because schistosome core I was six times more efficient as an acceptor than was the chitotetraose. In addition, schistosome core I formed more than one product. It is interesting that the blood type A oligosaccharide, which also has a GalNAc at the nonreducing end, was not a substrate.
The fucose-related ß-type iminosugars A and B were four- to sixfold more potent inhibitors of the schistosome fucosyltransferases in vitro than were the conventional -type iminosugars C and D. The iminosugars homofuconojirimycin and deoxyfuconojirimycin have been shown to be fucosyltransferase inhibitors with IC50 values of
70 mM (Qiao et al., 1996
). That study also demonstrated a synergistic effect between the inhibitors and GDP, with the potency increasing by 20- to 80-fold (IC50s
14 mM) in the presence of 30 µM GDP. This synergism is relevant to the use of iminosugars as inhibitors in vivo, because eukaryotic cells typically have an intracellular concentration of 1550 µM GDP. In in vivo experiments, iminosugar A blocked the expression of the epitope recognized by mAb 128C3/3 at a concentration of 5 µM, whereas its IC50 in vitro was 15 mM, suggesting that the synergism may be occurring in live schistosomes. The inhibitory effects of iminosugars on the expression of fucosylated structures may also be the result of effects on other fucose-metabolizing enzymes, such as fucosidases or those involved in the biosynthesis of GDP-Fuc. Although the compounds were not toxic to the adult worms in vitro, they may affect the parasite life cycle in vivo.
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Materials and methods |
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Preparation of S. mansoni extracts
The schistosome homogenates were prepared according to DeBose-Boyd et al. (1996) with minor modifications. Adult worms, cercariae, and eggs of S. mansoni were washed in cacodylate buffer (50 mM, pH 7.0) and sonicated for 10 s at 50% of the maximum output in a cell disrupter (Branson Sonic Power Co.). The homogenates were made to 1% in Triton X-100 and incubated on ice for 30 min, then centrifuged for 10 min at 15,000 r.p.m. at 4°C. The resulting supernatants were divided in aliquots and stored at 80°C. The protein concentration of each extract was determined by the BCA protein assay (Pierce, Rockford, IL) according to manufacturers protocol.
Materials
ANTS was obtained from Molecular Probes (Eugene, OR) as its disodium salt. Oligosaccharides were obtained from Oxford Glyco Systems (Oxford, UK), V-Labs (Convington, LA), Glycotech (Rockville, MD), or Calbiochem (La Jolla, CA), as presented in Table I. The electrophoretic reagents were obtained from Bio-Rad (Hercules, CA). Guanosine diphospho-[U14C]fucose, (GDP-fucose), ammonium salt (310 mCi/mmol) was obtained from Amersham Life Science (Piscataway, NJ). The imaging plates BAS-TR2040 were purchased from Fuji Photo Film (Tokyo). All other reagents were reagent grade.
Oligosaccharide synthesis
Synthesis of Sialyl2,3-LNnT. The biosynthesis of oligosaccharide NeuAc
2-3Galß1-4GlcNAcß1-3Galß1-4Glc, sialyl
2,3-LNnT (SLNnT) was performed with rat
2,3-(N)-sialyltransferase (Calbiochem). The enzyme (10 mU) was incubated with 800 nmoles of LNnT and 4 µmoles of CMP-ß-D-sialic acid (Calbiochem), in 50 mM MOPS, pH 7.4, with 0.2% Triton CF-54 and 0.5 mg/ml of ultrapure BSA (Boehringer Mannheim, Indianapolis, IN), a total volume of 160 µl for 6 h at 37°C. The reaction product was passed through an anion exchange column QAE Sephadex® A-50 (Pharmacia), and the sialylated sugars were eluted with 1 M ammonium formate, then lyophilized and resuspended in distilled water. A separate reaction mixture was also prepared using 0.1 mU of enzyme, 5 nmoles of LNnT, and 0.34 nmoles of CMP-ß-D-[U14C]sialic acid (300 mCi/mmol) (Amersham Life Science) in 10 µl and incubated under the same conditions. The sialylated oligosaccharide product was characterized by treatment with 1 mU of Newcastle disease virus neuraminidase (Boehringer Mannheim) for 1 h according to the manufacturers protocol. The purified products of the reactions were analyzed and the presence of sialic acid confirmed using FACE (data not shown).
Synthesis of schistosome core I. The biosynthesis of the oligosaccharide GalNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc, schistosome core I, was performed through adaptations of the method described by Palcic and Hindsgaul (1991). The reaction mixture was prepared as follows: 4.77 µmoles of N, N', N''-triacetylchitotriose (Calbiochem) was incubated with 15.3 µmoles of UDP-
-D-N-acetylgalactosamine (Calbiochem) and 6 U of ß1,4-galactosyltransferase from bovine milk, which has been demonstrated to form GalNAcß1,4GlcNAc linkages (Calbiochem) in 100 mM sodium cacodylate buffer, pH 7.5, and 0.5 mM MnCl2 at 37°C in a volume of 1.9 ml for 24 h, after which an additional 15.3 µmoles of UDP-
-D-N-acetylgalactosamine and 3 U of ß1,4-galactosyltransferase were added for final volume of 2.4 ml and the reaction was incubated for an additional 24 h. Finally, 7 U of alkaline phosphatase from calf intestine (Boehringer Mannheim) was added to the reaction, and the MnCl2 was adjusted to 5 mM and incubated for an other 48 h. The reaction rate was determined by removing a sample of the mixture every 24 h and submitting it to FACE analysis. The reaction was continued until it reached 95% completion. The ß1,4-galactosyltransferase reaction product presented a migration profile distinct from that of the starting material. The migration profile of the oligosaccharide product, schistosome core I, was consistent with the migration profile of chitotetraose (GlcNAcß1,4GlcNAcß1,4 GlcNAcß1,4 GlcNAc), which has identical mass and charge (data not shown).
Fluorescent labeling
All oligosaccharides used for the fucosyltransferase assay were prelabeled with ANTS as follows (Jackson, 1990, 1996): 100 nmoles of each oligosaccharide in water was placed in individual tubes and lyophilized in a centrifugal vacuum evaporator. To each dry sample was added 5 µl of 0.2 M ANTS in acetic acid/water (3:17, v/v) and 5 µl of 1.0 M NaCNBH3 solution in DMSO. The contents were mixed and centrifuged, incubated at 37°C for 15 h, and then dried under vacuum at 45°C for 4 h. Finally, the ANTS-labeled oligosaccharides were resuspended in water at 10 nmoles/µl and stored at 80°C until use. The fluorescent intensities of the labeled oligosaccharides were compared to a standard ANTS labeled glucose ladder (Bio-Rad), revealing that each oligosaccharide was >95% converted to its ANTS derivative (data not shown).
FACE fucosyltransferase assay
Fucosyltransferase reactions were performed in a 10-µl reaction volume containing 100 mM cacodylate buffer (pH 7.0), 5 mM ATP, 10 mM MnCl2, 50 µg extract protein from cercariae or adult worms or 16 µg protein from egg homogenates, 0.5 mM of ANTS-labeled acceptor (unless otherwise indicated), and 16.6 µM (115,000 c.p.m.) GDP-[14C]-Fuc (310 mCi/mmol). The reaction mixture was incubated at 37°C for times optimized for each reaction. At the end of the incubation the reaction was stopped by the addition of the electrophoresis loading buffer and the mixture was stored at 20°C prior to FACE analysis.
FACE analysis
The carbohydrate gel electrophoretic procedure was adapted from the protocol described by Jackson (1990, 1996). The fucosyltransferase reaction mixtures were subjected to PAGE using a Power PAC 3000 power supply (Bio-Rad) and a Mini-protean® II Cell. The electrophoretic buffer used was based on the Tris/HCl/glycine discontinuous system of Laemmli, without SDS. The polyacrylamide gels consisted of 32% (w/v) acrylamide and 2.0% bis-N,N'-methylenebisacrylamide as a cross-linker. The polymerization was initiated by addition of 8 µl of 5% ammonium persulfate solution and 4 µl N, N, N', N'-tetramethylethylenediamine per 5 ml gel solution. The samples were electrophoresed at 20 mA constant current at approximately 0°C. At the end of the electrophoresis fluorographic images were captured using a Kodak digital camera DC120 Zoom, with UV transillumination or by using the FACE Imager (Glyko, Novato, CA). The gels were then dried in a gel drier at 80°C for 1 h and exposed to an imaging plate (BAS-TR2040 phosphorimager plate; Fuji) for 3 days or to film (Biomax MR; Kodak, Rochester, NY) to quantify radioactivity.
Quantification of [14C] fucose
The dried gels were exposed to the imaging plates simultaneously with an autoradiographic [14C] micro-scale (Amersham Life Science), ranging from 0.045 to 45 pCi (approx. 0.1100 d.p.m.). The imaging plates were analyzed using a BAS 1000 phosphorimager (Fuji). The pixel density of the standards was determined using MacBAS 2.5 software (Fuji) and plotted against d.p.m., and a correlation curve was determined by linear regression using StatView 4.5 (Abacus Concepts, Berkeley, CA). The correlation curve was then used to the convert the pixel densities of the fucosyltransferase product bands to d.p.m.
Fucosidase assay
Fucosidase assays were performed in 10 µl of 100 mM cacodylate buffer, pH 5.0, containing 5 nmoles of the ANTS-labeled substrate oligosaccharide 3'F-Lac (Galß1,4(Fuc1,3)Glc-ANTS) or 2'F-Lac (Fuc
1,2Galß1,4Glc-ANTS), which form lactose (Galß1,4Glc-ANTS) after cleavage of the attached fucose. The reaction mixtures were incubated at 37°C in the presence of 50 µg protein from cercariae or adult worms or 16 µg protein from egg homogenates. The reaction was stopped by addition of 2.5 µl 5x gel electrophoresis sample buffer, and the reaction products were analyzed by FACE. The migration pattern of the ANTS-labeled lactose product of the fucosidase reactions on FACE was distinct from that of the substrates 3'F-Lac and 2'F-Lac. The rate of product formation was determined in triplicate by quantifying the fluorescence of each of the bands corresponding to the substrates and products and comparing these to standards, using Glyko FACE Analytical Software (Glyko).
Iminosugars
A new class of iminosugars, 1-N-iminosugars, was designed as described previously (Ichikawa et al., 1998). These 1-N-iminosugars contain a nitrogen atom in the anomeric position, whereas the conventional deoxynojirimycin-type iminosugars have a nitrogen atom in place of the ring oxygen of the monosaccharide (Figure 5A) (Ichikawa et al., 1998
). The 1-N-iminosugars are more potent inhibitors of ß-glycosidases than the deoxynojirimicyn-type iminosugars, which are potent inhibitors of
-glycosidases (Ichikawa et al., 1998
). The iminosugars used in this study were designated iminosugar A, iminosugar B, and iminosugar C (Figure 5B). Iminosugar D (deoxyfuconojirimycin) was purchased from Calbiochem.
Radioisotopic labeling of adult worms with [35S] methionine
Adult worms were cultured in vitro for 48 h in the presence or absence of iminosugar A at concentrations of 5500 µM in DMEM with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES buffer (pH 7.5), 0.375% sodium bicarbonate, 0.2 U/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. The worms (three males and three females per well) were cultured in 24-well plates, 0.5 ml medium, at 37°C under a 5% CO2 atmosphere using duplicate wells for each condition. The medium and the iminosugars were replaced every 24 h. After 48 h the worms were metabolically labeled with [35S]methionine (600 Ci/mmol, 0.6 mCi/ml, Amersham) for 18 h in methionine-free DMEM plus the above supplements. At the end of the labeling period the worms were washed with DMEM and solubilized.
Radioimmunopreciptation
Tegumental membranes of [35S]methionine-labeled worms were solubilized with 0.2% Triton X-100, 10 mM CaCl2, 50 mM TrisHCl (pH 7.8), and protease inhibitor cocktail (Complete EDTA-free, Boehringer Mannheim) for 30 min on ice. An aliquot of each labeled extract was then precipitated with trichloroacetic acid and the total radioactivity measured in triplicate. The labeled extracts were immunoprecipitated by standard protocols (Norden and Strand, 1985) with mAb 128C3/3 [which recognizes a highly fucosyltated structure described by Levery et al. (1992)
] and mAb 103A3 (used as a negative control).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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