Glycobiology Advance Access originally published online on January 20, 2006
Glycobiology 2006 16(5):431-439; doi:10.1093/glycob/cwj079
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A specific detection of GlcNAcß1-6Man
1 branches in N-linked glycoproteins based on the specificity of N-acetylglucosaminyltransferase VI
2 Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan and 3 Department of Molecular Genetics, Kochi University Medical School, Kochi 783-8505, Japan
1 To whom correspondence should be addressed; e-mail: tom_taguchi{at}biochem.med.osaka-u.ac.jp
Received on December 1, 2005; revised on January 12, 2006; accepted on January 15, 2006
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Abstract |
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Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface—in particular, the production of GlcNAcß1-6Man
1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5'-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcß1-6Man1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto 1-acid glycoprotein (AGP), which is known to contain GlcNAcß1-6Man1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcß1-6Man1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and ß-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS–PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L4-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L4-PHA.
Key words:
1-acid glycoprotein
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cancer cells
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GlcNAcß1-6Man1 branch
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GnT V
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GnT VI
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementEnzymeAcknowledgmentsReferences |
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It is well known that malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface (Warren et al., 1972
; Van Beek et al., 1975; Warren et al., 1978; Santer and Glick, 1979; Santer et al., 1984; Collard et al., 1985), in particular, the presence of ß1-6GlcNAc-branching N-linked glycans with poly-N-acetyllactosamine structures (Yamashita et al., 1984; Pierce and Arango, 1986). The enzyme responsible for generating ß1–6GlcNAc-branching N-linked glycans has been purified and cloned (Gu et al., 1993; Shoreibah et al., 1993). The activity of this enzyme, uridine (5'-)diphosphate (UDP)-N-acetylglucosamine: -6-D-mannoside ß1-6N-acetylglucosaminyltransferase (GnT V), becomes elevated in fibroblast and epithelial cell lines when oncogenes are expressed (v-src, T24-H-ras) in cells infected with polyoma virus or Rous sarcoma virus (Yamashita et al., 1985; Dennis et al., 1987, 1989; Arango and Pierce, 1988) and in an early phase of hepatocarcinogenesis (Ito et al., 2001). It is noteworthy that the transcription of the GnT V gene is positively regulated by these oncogenes (Kang et al., 1996; Chen et al., 1998). Forced expression of the GnT V gene in epithelial cells results in the loss of contact inhibition, increased cell motility, morphological transformations in culture, and tumor formation in athymic nude mice (Demetriou et al., 1995). In another experiment, involving the overexpression of the GnT V gene in a gastric cancer cell line, an increase in the half-life of matriptase was found, which could play an important role in the prometastatic stage of cancer cells (Ihara et al., 2002, 2004). More conclusive insights into GnT V function in vivo were directly obtained from GnT V-deficient mice, which exhibit suppression in tumor growth and metastasis (Granovsky et al., 2000; Dennis et al., 2002) and negative regulation of T-cell activation and autoimmunity (Demetriou et al., 2001), although the identification of glycoproteins that are modified by the action of GnT V remains incomplete. To date, the plant lectin leukoagglutinating phytohemagglutinin (L4-PHA) is the sole reagent that exhibits reactivity toward ß1-6GlcNAc-branching N-glycans (Cummings and Kornfeld 1982a, 1982b). Interestingly, only a subset of N-linked glycoproteins appears to serveas a GnT V target in this lectin precipitation assay (Ochwat et al., 2004).
We succeeded in purifying and cloning UDP-GlcNAc: GlcNAcß1-6(GlcNAcß1-2)Man1-R[GlcNAc to Man]-ß1, 4-N-acetylglucosaminyltransferase VI (GnT VI) (Taguchi et al., 1997, 1998, 2000; Sakamoto et al., 2000). This enzyme has a strict requirement for the GlcNAcß1-6Man1 structure on the acceptor substrate for its activity (Taguchi et al., 2000). By exploiting this substrate specificity, which was characterized only using pyridylaminated free N-glycans, we sought to establish a method for detecting GlcNAcß1-6Man1 branch in N-linked glycoproteins (Scheme 1). Human asialo-agalacto 1-acid glycoprotein (AGP), which is known to contain GlcNAcß1-6Man1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI and [14C]-UDP-GlcNAc, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcß1-6Man1 structure, were not. Even with crude materials such as human serum, this method permitted a few proteins, one of which appeared to be AGP, to be detected. Furthermore, this method could detect a protein, which was also positively stained with L4-PHA, using glycoproteins prepared from WiDr human colon cancer cells. By using glycoproteins prepared from B16F1 mouse melanoma cells, this method clearly detected two bands that were not visible with L4-PHA. This method should provide a useful complement to the current method, which relies on the specificity of L4-PHA.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementEnzymeAcknowledgmentsReferences |
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Purification of soluble His-tagged GnT VI secreted from sf 21 cell
A baculovirus/insect cell expression system was used to prepare recombinant GnT VI. A soluble form of GnT VI was designed by deletion of its tentative transmembrane domain and the addition of a poly His-tag at the N-terminus. The activity of the recombinant GnT VI was assayed using the fluorescently labeled agalacto triantennary glycan [2,(2,6)]-2-aminopyridine ([2,(2,6)]-PA) as an acceptor substrate, basically following the method originally described (Taguchi et al., 2000
). An aliquot of 200 ml of conditioned medium from transfected sf21 cells was collected and used for purification. The majority of the GnT VI activity (around 60%) was recovered in the fraction precipitated with 50% saturated ammonium sulfate. Although a portion of the GnT VI activity passed through Ni2+-chelating Sepharose FF column (probably due to cleavage of the His-tag), a >200-fold purification was achieved in this step. The specific activity of this fraction was 12,900 pmol/h/mg. This enzyme fraction was then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed by silver staining and by Western blotting with antipoly His-tag antibody. The band appeared at 58 kDa (Figure 1A) was confirmed to be recombinant GnT VI, with the result of Western blotting (Figure 1B). This size was consistent with the estimated molecular weight. The high-performance liquid chromatography profile obtained with this enzyme fraction is shown in Figure 2. In the presence of UDP-GlcNAc (Figure 2A), only one additional peak appeared, with a retention time of 14.7 min, compared with the reaction without UDP-GlcNAc (Figure 2B). This peak was previously assigned to [2,(2,4,6)]-PA, thus confirming the specificity of the enzyme. Moreover, no detectable GnT III, IV, or V activity was found with agalacto biantennary glycan ([2,2]-PA) as an acceptor substrate (data not shown). This fraction was used as an enzyme source throughout this study.
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Preparation of acceptor substrates from purified proteins
To establish an assay for detecting GlcNAcß1-6Man1 branches in N-linked glycoproteins, three glycoproteins (AGP, Tfn, and fetuin) were chosen as model substrates, since the structures of their N-linked glycans are well known.
Human AGP is composed of 201 amino acids (44 kDa) and contains five potential N-glycosylation sites. According to a previous report (Shiyan and Bovin, 1997), almost half of its N-glycans possess a GlcNAcß1-6Man1 branch. Thus, human AGP represents a good positive control for this novel assay with GnT VI.
Human Tfn contains two potential N-glycosylation sites, and the major structures of its N-glycans are well known to be biantennary (Spik et al., 1975). Bovine fetuin contains three potential N-glycosylation sites. It was previously shown that one N-glycan is biantennary and the other two are triantennary which do not contain GlcNAcß1-6Man1 branches (Green et al., 1988). Thus, both Tfn and fetuin were used as negative controls in evaluating this assay.
Since GnT VI cannot act on galactosylated glycans (T.Taguchi, unpublished data), the three glycoproteins were first digested extensively with sialidase and ß-galactosidase to expose internal GlcNAc residues linked to -Man. The extent of desialylation and degalactosylation was examined by means of a lectin blotting using RCA120. This lectin is known to detect nonreducing terminal ß-Gal residues. All of the three glycoproteins, after sialidase and ß-galactosidase digestion, were not detected by RCA120 (Figure 3A: lanes 3, 6, and 9), whereas the desialylated glycoproteins were clearly visualized with this lectin (Figure 3A: lanes 2, 5, and 8). This result, in conjunction with the change in their mobility on SDS–PAGE (Figure 3B), demonstrates that the glycosidase treatments were complete.
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GnT VI reaction with asialo-agalacto purified glycoproteins
Asialo-agalacto glycoproteins (AGP, Tfn, and fetuin) were then examined as to whether they function as acceptor substrates for recombinant GnT VI. These glycoproteins were incubated with recombinant GnT VI in the presence of radiolabeled UDP-GlcNAc. After the reaction, the proteins were precipitated with methanol/chloroform, and the resulting pellet was solubilized in SDS. After separation by 10% SDS–PAGE gel, the gel was autoradiographed with a BAS system. As shown in Figure 4, only human asialo-agalacto AGP was detected at around 40 kDa (lane 3), whereas the other proteins were not. Intact and asialo AGP were found not to function as acceptor substrates (lanes 1 and 2). These results show that only asialo-agalacto glycoproteins with GlcNAcß1-6Man1 branches are able to serve as substrates for GnT VI.
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Preparation of asialo-agalacto serum glycoproteins
Since we confirmed that GnT VI recognized purified glycoproteins with GlcNAcß1-6Man1 branches, we examined the possibility that this assay could be extended to crude materials. We chose human serum, since we already knew that AGP in human serum functioned as a positive control in this method.
After sialidase and ß-galactosidase digestion, silver staining and lectin blotting with RCA120 were performed. As shown in Figure 5B, degalactosylation appeared to proceed well, since most of the staining observed in lane 2 was absent in lane 3 except for one band at around 55 kDa. This was also confirmed by the blotting of AGP in human serum (Figure 5C). Through desialylation and degalactosylation processes, the molecular weight of AGP gradually became smaller. The final product (Figure 5C: lane 3) ran at around 40 kDa, close to that for the purified AGP (Figure 3B: lane 3), confirming that the enzyme treatment was complete.
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GnT VI reaction with asialo-agalacto serum glycoproteins
Asialo-agalacto serum glycoproteins were then examined as to whether they could function as acceptor substrates for recombinant GnT VI. As shown in Figure 5D, human serum that had been pretreated with sialidase and ß-galactosidase gave several radioactive bands (lane 3). Human serum without enzyme treatment (lane 1) or pretreated with sialidase (lane 2) was not found to function as acceptor substrates. The negative control (without recombinant GnT VI) did not give any band (data not shown). The radioactive band at around 40 kDa was assumed to be asialo-agalacto AGP, judging from its molecular weight (Figure 5C: lane 3). The identity of the other bands has not yet been confirmed.
Preparation of asialo-agalacto glycoproteins from cancer cells
Next we wanted to ask if this method could be applied to glycoproteins prepared from tissue culture cells and also wanted to compare this method to L4-PHA staining in terms of specificity and sensitivity. Since the relationship between tumorigenesis and appearance of GlcNAcß1-6Man1 branch has been proposed, we chose tumor cells (WiDr, human colon cancer cells; B16F1, mouse melanoma cells) as model. After sialidase and ß-galactosidase digestion, silver staining and lectin blotting with either RCA120 or L4-PHA were performed. As shown in Figure 6B (WiDr) and Figure 7B (B16F1), degalactosylation was confirmed with lectin blotting using RCA120, since most of the staining observed in lane 2 was absent in lane 3. As shown in Figure 6C (WiDr), one major band was detected with lectin blotting using L4-PHA (lanes 2 and 3). As shown in Figure 7C (B16F1), a doublet band was detected using sample pretreated with sialidase (lane 2), and it disappeared after sialidase and ß-galactosidase treatment (lane 3).
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GnT VI reaction with glycoproteins from tumor cells
Asialo-agalacto glycoproteins prepared from WiDr were then examined as to whether they could function as acceptor substrates for recombinant GnT VI. As shown in Figure 6D (lane 3), only one band was detected at around 110 kDa, close to that detected with L4-PHA (Figure 6C, lane 3). In the case of B16F1 cell, two major bands were detected at around 90 and 60 kDa (Figure 7D, lane 3).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementEnzymeAcknowledgmentsReferences |
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A method for identifying GlcNAcß1-6Man
1 branches in N-linked glycoproteins using recombinant GnT VI and radiolabeled UDP-GlcNAc was evaluated. This assay exploits the fact that GnT VI has a strict requirement for GlcNAcß1-6Man1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. The aim of this study was largely fulfilled. As shown in Figure 4, human asialo-agalacto AGP that is known to contain GlcNAcß1-6Man1 branches in its N-linked glycan chains was radiolabeled with GnT VI, whereas human asialo-agalacto Tfn and bovine fetuin, neither of which contains a GlcNAcß1-6Man1 structure, were not. Even with crude materials such as human serum, this method successfully detected several specific glycoproteins, one of which was assumed to be AGP, judging from its mobility shift on SDS–PAGE gels (Figure 5D).
It is well known that malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface, in particular, the production of GlcNAcß1-6Man1 structures with poly-N-acetyllactosamine sequences. So far, L4-PHA has been the sole available reagent for detecting GlcNAcß1-6Man1 branches on N-glycans/N-linked glycoproteins. So, we tested whether our enzymatic method is comparable to the one with L4-PHA lectin using tumor cell lines (WiDr human colon cancer cell and B16F1 mouse melanoma cell).
As shown in Figure 6C, one major band at around 110 kDa was detected with L4-PHA after sialidase or sialidase/ß-galactosidase treatment of glycoproteins prepared from WiDr cells. Importantly, the size of this band was close to the one detected with GnT VI reaction (Figure 6D, lane 3), corroborating the specificity of our method. Interesting result was obtained from B16F1 cells. As shown in Figure 7C, one doublet at around 75 kDa was detected with L4-PHA (sialidase treated, lane 2), whereas two discrete bands (90 and 60 kDa) were detected with GnT VI reaction (Figure 7D, lane 3). This result suggests that two independent methods detected different proteins, although direct comparison is rather difficult in this case, as glycoproteins from B16F1 cells which were pretreated with sialidase/ß-galactosidase did not yield any good band with L4-PHA lectin (Figure 7C, lane 3).
This issue might be relevant to the steric hindrance effects of protein moiety against the carbohydrate chains. The effects could explain the fact that L4-PHA agarose column was not able to retain human AGP proteins under a variety of conditions (data not shown). To relieve this, a future challenge will be the limited digestion of N-linked glycoproteins by a protease before a reaction with recombinant GnT VI. This would also make the sialidase and ß-galactosidase pretreatment, used in the preparation of substrates for GnT VI reaction, to be more efficient.
In summary, an enzymatic assay for identifying GlcNAcß1-6Man1 branches in N-linked glycoproteins is described. This method should provide a useful complement to the current method, which relies on the specificity of L4-PHA.
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Materials and Methods |
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Materials
All materials were purchased from the following suppliers: ammonium sulfate, imidazol, UDP-GlcNAc, GlcNAc, human AGP, Tfn, and fetuin derived from fetal calf serum were from Sigma (St. Louis, MO, USA); Ni2+-chelating Sepharose FF, uridine diphospho-N-acetyl-[14C]-glucosamine, and enhanced chemiluminescence (ECL) from Amersham (Uppsala, Sweden); Tris, NaCl, HEPES, MES, MOPS, and neuraminidase (Arthrobacter ureafaciens) from Nacalai Tesque (Kyoto, Japan); ß-galactosidase (Jack bean), L4-PHA-biotin, RCA120-biotin (CAS172304-66-4), L4-PHA agarose from Seikagaku Corporation (Tokyo, Japan), BaculoGold from BD Biosciences (San Jose, CA, USA), Lipofectin from Invitrogen (Tokyo, Japan); dialysis tube (seamless cellulose tubing small size 30) from Wako (Osaka, Japan); Big Dye Terminator cycle sequencing kit from PerkinElmer Life Science (Boston, MA, USA); silver staining kit from Daiichi Pure Chemicals (Tokyo, Japan).
Construction of a vector encoding GnT VI-pAcGP67H
To prepare a soluble His6-tagged (at N-terminus) chicken GnT VI lacking the transmembrane domain, the full-length cDNA was used as a template in a PCR with the following primers: 5'-GACGAATTCCTCCTCCTTCTGCACAG-3' and 5'-GACAGATCTTCAGGTGCCTGCAGTCC-3'. The amplified product was then subcloned into pBlueScript (Stratagene, La Jolla, CA, USA). Sequencing was carried out with Big Dye Terminator cycle sequencing kit with a DNA sequencer (Applied Biosystems model 377). For transfection, EcoRI and BglII sites were used to ligate into vaculovirus vector pAcGP67H, which was modified to have His-tag sequence (Pharmingen, San Jose, CA, USA). The plasmid of GnT VI-pAcGP67H was then transfected into sf21 cells for protein purification.
Purification of recombinant GnT VI
An aliquot of 200 ml of conditioned medium recovered from sf21 cells was first treated with 30% saturated ammonium sulfate solution for 1 h. After centrifugation (15 min, 10 000 gmax), the supernatant was recovered and adjusted to a 50% saturated ammonium sulfate solution. The resulting pellet was dialyzed for 2 days against 50 mM HEPES (pH 7.5)/0.4 M NaCl/20% glycerol (buffer A). An aliquot of 5 ml of Ni2+-chelating Sepharose FF resin was layered on the 1 mL of chelating Sepharose FF resin without metal ions to prevent any possible leakage of Ni2+ from the column. The dialyzed sample was applied directly to a column of Ni2+-chelating Sepharose FF that had been equilibrated with buffer A. The column was washed with 25 mL of buffer A, followed with 10 mL of 0.1 M imidazol/buffer A and 5 mL of 0.25 M imidazol/buffer A. GnT VI activity bound to the column was largely eluted with another round of washing with 5 mL of 0.25 M imidazol/buffer A.
Preparation of [2,(2,6)]-PA from biantennary sugar chain
Asialo-agalacto biantennary sugar chain [2,2]-PA was reacted with the conditioned medium of sf21 cells, which express soluble GnT V. The reaction was carried out at 37°C overnight in a total volume of 500 µL with the following components: 160 mM HEPES (pH 8.0), 10 mM UDP-GlcNAc, 90 mM GlcNAc, 0.6% Triton X-100, 100 pmol of [2,2]-PA, and 10 µL of GnT V enzyme fraction. The reaction product [2,(2,6)]-PA was purified with TSK-Gel ODS-80TM column (150 x 4.6 mm; Tosoh, Tokyo, Japan) at 55°C equilibrated with 20 mM ammonium acetate (pH 4.0) at a flow rate of 1.6 mL/min. Fluorescence was monitored with excitation and emission wavelengths of 320 and 400 nm, respectively. The fraction containing the product [2,(2,6)]-PA was pooled and concentrated on a rotary evaporator. GnT VI activity was assayed with [2,(2,6)]-PA, as described previously (Taguchi et al., 2000).
Glycosidases treatment of glycoproteins
An aliquot of 20 µg of AGP, Tfn, and fetuin was digested with 1 mU of neuraminidase for 3 h and 100 mU of ß-galactosidase overnight in the 200 mM MES buffer (pH 5.5) at 37°C. In the case of human total serum proteins, 10 µL of serum was digested with 10 mU sialidase and 1250 mU ß-galactosidase in the 200 mM MES buffer (pH 5.5)/1 mM DTT/protease inhibitors (Complete EDTA free, Roche, Germany) overnight at 37°C. In the case of glycoproteins prepared from WiDr and B16F1 cells, cell pellets from WiDr and B16F1 were lysed in 100 mM MES buffer (pH 6.5)/1 mM DTT/protease inhibitors/1% TritonX-100 overnight at 4°C with rotation. After ultracentrifugation at 45 000 gmax for 1 h at 4°C, the supernatant (containing 500 µg protein) was carefully removed and then subjected to digestion with 20 mU sialidase and 250 mU ß-galactosidase overnight at 37°C.
Gel electrophoresis
Electrophoresis was performed on a 10% SDS–PAGE under reducing conditions (Laemmli, 1970). Silver staining was performed using silver staining kit. Lectin blotting was performed with 1 µg/mL of biotinylated RCA120 or biotinylated L4-PHA. An anti-human AGP antibody was used at a final concentration of 10 µg/mL. An antipoly His-tag antibody was used at final concentration of 0.05 µg/mL. The washing and developing process with ECL were performed according to the manufacture’s instructions.
GnT VI reaction with [14C]-UDP-GlcNAc
An aliquot of 1 µg of control purified glycoproteins, 100 µg of human serum proteins, or 100 µg of glycoproteins prepared from cancer cell lines (WiDr or B16F1) that had been digested with sialidase and ß-galactosidase were used in the GnT VI reaction. The incubation mixture contained the following components: 160 mM HEPES (pH 8.0), 0.1 mCi [14C]-UDP-GlcNAc, 90 mM GlcNAc, 30 mM MnCl2, 0.6% Triton X-100, and 5 µL of GnT VI enzyme fraction. The total volume of the reaction was 20 µL for purified glycoproteins, 30 µL for serum proteins, and 35 µL for glycoproteins from cancer cells. After incubation for 5 h at 37°C, 1 mL of methanol : chloroform (1 : 2) was added and mixed. After centrifugation at 15 000 g for 15 min at room temperature, the precipitate was recovered and subjected to SDS–PAGE (10% gel). Radiolabeled materials were then detected by BAS2500 (Fujifilm, Tokyo, Japan).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementEnzymeAcknowledgmentsReferences |
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None declared.
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Enzyme |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementEnzymeAcknowledgmentsReferences |
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N-acetylglucosaminyltransferase VI (EC 2.4.1.201 [EC] ).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementEnzymeAcknowledgmentsReferences |
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This work was supported, in part, by The 21st Century Center of Excellence Program from the Ministry of Education, Science, Culture, Sports, and Technology of Japan.
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Abbreviations |
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AGP,
1-acid glycoprotein; ECL, enhanced chemiluminescence; GnT V, N-acetylglucosaminyltransferase V; GlcNAc, N-acetyl-D-glucosamine; GnT VI, N-acetylglucosaminyltransferase VI; L4-PHA, leukoagglutinating phytohemagglutinin; PA, 2-aminopyridine; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tfn, transferrin; UDP, uridine (5'-)diphosphate; [2,2]-PA, GlcNAcß1-2Man1-3(GlcNAcß1–2Man1-6)Manß1-4GlcNAcß1-4GlcNAc-PA; [2,(2,6)]-PA, GlcNAcß1-2Man1-3(GlcNAcß1-2(GlcNAcß1-6)Man1-6)Manß1-4GlcNAcß1-4GlcNAc-PA; [2,(2,4,6)]-PA, GlcNAcß1-2Man1-3(GlcNAcß1-2(GlcNAcß1-4)(GlcNAcß1-6)Man1-6)Manß1-4GlcNAcß1-4GlcNAc-PA |
References |
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