Glycobiology Advance Access originally published online on February 23, 2005
Glycobiology 2005 15(7):7C-13C; doi:10.1093/glycob/cwi050
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COMMUNICATION |
Expression of N-acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs)-1 and -4 in human monocytes: GlcNAc6ST-1 is implicated in the generation of the 6-sulfo N-acetyllactosamine/Lewis x epitope on CD44 and is induced by TNF-
2 Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3; and 3 Program of Experimental Pathology, Aichi Cancer Center, Nagoya 464, Japan
1 To whom correspondence should be addressed; e-mail: pauline{at}interchange.ubc.ca
Received on December 16, 2004; revised on February 10, 2005; accepted on February 15, 2005
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Abstract |
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Sulfation at the 6-O position of N-acetylglucosamine (GlcNAc) in the context of sialyl 6-sulfo Lewis x occurs constitutively on specific glycoproteins present on high-walled endothelial venules (HEV) and is important for L-selectin dependent homing of lymphocytes. Here, the proinflammatory cytokine, TNF-
, induced the expression of 6-sulfo N-acetyllactosamine (LacNAc)/Lewis x on human peripheral blood monocytes (PBM). This epitope was detected by monoclonal antibody (mAb) AG107 after neuraminidase treatment suggesting a sialylated epitope, which was present on the cell adhesion molecule, CD44. Treatment of human PBM with TNF- up-regulated the expression of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) and GlcNAc6ST-4, as determined by reverse transcriptase polymerase chain reaction (RT–PCR). However, only GlcNAc6ST-1 was induced by TNF- in the human SR91 cell line, which also up-regulated the AG107 epitope. In ECV304 cells, the expression of GlcNAc6ST-4 alone was insufficient to generate the AG107 epitope. However, the transfection of GlcNAc6ST-1 resulted in significant sulfate incorporation into CD44 and generated the 6-sulfo LacNAc/Lewis x epitope on CD44, which was present largely on N-linked glycans. This demonstrates the induction of GlcNAc6STs in human monocytes in response to TNF- and implicates GlcNAc6ST-1 in the generation of the 6-sulfo LacNAc/Lewis x epitope on CD44. Key words: carbohydrate sulfation / CD44 / inflammation / monocytes / sulfotransferase
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Introduction |
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The sulfation of proteoglycans and glycoproteins has been implicated in many biological processes including cell–cell adhesion, cell proliferation through the capture of soluble growth factors and chemokines and in viral and bacterial invasion (reviewed in Brockhausen and Kuhn, 1997
; Bowman and Bertozzi, 1999). Mucins are a heterogeneous family of sialylated and sulfated glycoproteins present in the human bronchial and intestinal mucosa where they play a protective role and contribute to innate immunity at these sites (reviewed in Lamblin et al., 2001). Glycoproteins on high-walled endothelial venules (HEV) present sialyl 6-sulfo Lewis x determinants that can mediate lymphocyte homing to lymph nodes by serving as ligands for L-selectin on circulating lymphocytes in mice (Hemmerich and Rosen, 2000; Hemmerich et al., 2001; van Zante et al., 2003). In chronic inflammation, HEV-like vessels are induced and L-selectin ligands are detected on the endothelial cells at the inflammatory site (reviewed in Rosen, 1999).
CD44 is an adhesion molecule that is expressed on various cell types and has been implicated in a variety of processes including tumor metastasis (reviewed in Herrlich et al., 1993), T lymphocyte recruitment to an inflammatory site (DeGrendele et al., 1997), and the resolution of an inflammatory response in the lung (Teder et al., 2002). CD44 is sulfated in human peripheral blood monocytes (PBM) (Brown et al., 2001) and in the human myelo-leukemic cell line, SR91 (Maiti et al., 1998). In both cases, CD44 sulfation was increased upon exposure to the proinflammatory cytokine, TNF-. Investigation into the sulfation of CD44 in SR91 cells revealed a complex pattern of chondroitin and glycan sulfation that was altered upon TNF- stimulation (Delcommenne et al., 2002). Experiments involving a series of monoclonal antibodies (mAbs) directed at different sulfated carbohydrate epitopes identified 6-sulfo N-acetyllactosamine (LacNAc)/Lewis x on CD44 that was unmasked after removal of terminal sialic acid residues with neuraminidase (Delcommenne et al., 2002). This suggested that the expression or activity of specific glycosyltransferases or N-acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs) might be induced by TNF- and responsible for the increased carbohydrate sulfation of CD44.
Five Golgi-resident GlcNAc6STs have been identified in humans (reviewed in Grunwell and Bertozzi, 2002). Of these, GlcNAc6ST-2, -3, and -5 show very specific, restricted expression to HEV, intestine, and cornea, respectively, whereas GlcNAc6ST-1 (Uchimura et al., 1998) and -4 (Kitagawa et al., 2000; Uchimura et al., 2000) are widely expressed. In GlcNAc6ST-2 knockout mice, there was a substantial loss of sulfated L-selectin ligands on HEV, which was associated with a 
50% reduction in lymphocyte homing to peripheral lymph nodes (Hemmerich et al., 2001). GlcNAc6ST-1 knockout mice develop normally but have reduced expression of sialyl 6-sulfo Lewis x in HEV and a 25% reduction in homing to peripheral lymph nodes (Uchimura et al., 2004). GlcNAc6ST-1 and -4 transcripts are present in peripheral blood leukocytes (Uchimura et al., 1998; Bhakta et al., 2000), but the expression within the different leukocyte populations is not known. In this study, GlcNAc6ST-1 and -4 transcripts were identified in human PBM and the expression of both sulfotransferases was up-regulated by TNF-. TNF- up-regulated the expression of 6-sulfo LacNAc/Lewis x, which was detected after neuraminidase treatment by the AG107 mAb (Izawa et al., 2000) and was present on the cell adhesion molecule, CD44. GlcNAc6ST-1 was implicated in the synthesis of the 6-sulfo LacNAc/Lewis x determinant on CD44, where it was expressed predominantly on N-linked glycans.
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Results |
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To determine if the increased CD44 sulfation observed in human PBM (Brown et al., 2001
) was associated with the increased expression of 6-sulfo LacNAc/Lewis x in response to TNF-, as had been observed for SR91 cells (Delcommenne et al., 2002), we treated unstimulated and TNF--stimulated human PBM (CD14+ peripheral blood mononuclear cells [PBMC]) with neuraminidase and incubated with the mAb AG107 that recognizes 6-sulfo LacNAc/Lewis x (Izawa et al., 2000). Flow cytometry of PBM demonstrated that unstimulated PBM expressed low levels of the AG107 epitope after neuraminidase treatment and that this was significantly increased after TNF- stimulation (Figure 1A). Similar data were also obtained with another mAb that also recognizes 6-sulfo LacNAc/Lewis x, AG105 (data not shown). A western blot with mAb AG107 of immunoprecipitated CD44 from unstimulated and TNF--stimulated PBM established that the sulfated determinant detected by AG107 was present on CD44 (Figure 1B). The expression of CD44 and the AG107 determinant increased upon TNF- stimulation. On average, the AG107 epitope increased 5.9 ± 2.9 (n = 3) fold per cell and showed a slight increase (2.0 ± 0.5 [n = 3] fold) per CD44 molecule. On the other hand, CD44 isolated from CD14– PBMC did not bind the AG107 mAb, even after treatment with TNF- (data not shown). This indicated that the AG107 epitope, 6-sulfo LacNAc/Lewis x, was up-regulated in human PBM in response to TNF- and was present on CD44. The lack of mAb AG107 reactivity before neuraminidase treatment indicated that the determinant present on human PBM was masked by sialic acid, suggesting that the sulfated epitope expressed on CD44 is sialyl 6-sulfo LacNAc/Lewis x.
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To determine if TNF- up-regulated the expression of the AG107 epitope, 6-sulfo LacNAc/Lewis x, by inducing the expression of a specific sulfotransferase, we investigated the transcript levels of all known GlcNAc6STs by semiquantitative reverse transcriptase polymerase chain reaction (RT–PCR) in SR91 cells and human PBM. GlcNAc6ST-4 was detected at low levels in SR91 cells and this level did not change upon TNF- stimulation (Figure 2A). In contrast, GlcNAc6ST-1 transcripts were not readily detectable in unstimulated SR91 cells, but were induced significantly upon TNF- stimulation (11.7 ± 5.1 [n = 10] fold). Transcripts for GlcNAc6ST-2, -3, or -5 were not detected or detected at low levels and were not increased upon TNF- stimulation (Figure 2A). This was also found to be the case for the galactose and galactose/N-acetylgalactosamine (Gal/GalNAc) 6-O-sulfotransferases, KSGal6ST and C6ST-1, respectively (data not shown). Analysis of the GlcNAc6STs present in CD14+ PBMC revealed that transcripts for GlcNAc6ST-1 and -4 were present at low levels and that the stimulation of the cells with TNF- increased the expression level of both transcripts, whereas transcripts for GlcNAc6ST-2, -3, and -5 were either not detected or present at very low levels and were not increased with TNF- stimulation (Figure 2B). Normalizing GlcNAc6ST transcript levels with that of ß-actin indicated that GlcNAc6ST-1 transcripts increased 2.0 ± 0.2 (n = 5) fold and GlcNAc6ST-4 transcripts increased 2.0 ± 0.6 (n = 5) fold in response to TNF-. These increases were specific to CD14+ PBMC as transcript levels for GlcNAc6ST-1 and -4 were similar in unstimulated and TNF--stimulated CD14– PBMC (data not shown). These results suggest that in CD14+ PBMC, the expression of 6-sulfo LacNAc/Lewis x on CD44 may be regulated by the activities of GlcNAc6ST-1, -4, or both, whereas the results from the SR91 cells suggest that GlcNAc6ST-1 is the enzyme involved in generating the inducible 6-sulfo LacNAc/Lewis x determinant on CD44. A time course of TNF- stimulation on the SR91 cells revealed that GlcNAc6ST-1 transcript levels began to increase as early as 1 h after exposure to TNF- (Figure 2C).
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To ascertain whether GlcNAc6ST-1 and/or -4 contribute towards the generation of the 6-sulfo LacNAc/Lewis x epitope on CD44, we examined ECV304 cells or ECV304 cells transfected with GlcNAc6ST-1, 1,3 fucosyltransferase VII (Fuc-T VII), or both (Kimura et al., 1999). RT–PCR revealed that ECV304 cells expressed significant levels of transcripts for GlcNAc6ST-4, but did not express significant levels of transcripts for GlcNAc6ST-1, -2, -3, and -5 (Figure 3A and data not shown). As expected, GlcNAc6ST-1 transfected ECV304 cells expressed transcripts for both GlcNAc6ST-1 and -4 (Figure 3A). To distinguish which sulfotransferase was necessary for CD44 sulfation, we immunoprecipitated CD44 from [35S]sulfate-labeled ECV304 and GlcNAc6ST-1 transfected ECV304 cells. As shown in Figure 3(B), CD44 from ECV304 cells incorporated very low levels of [35S]sulfate whereas CD44 from GlcNAc6ST-1 transfected cells incorporated significantly higher levels of [35S]sulfate (5.7 ± 1.7 [n = 3] fold). This shows that the expression of GlcNAc6ST-1 leads to the sulfation of CD44.
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To determine if this GlcNAc6ST-1 dependent sulfation of CD44 resulted in the generation of the 6-sulfo LacNAc/Lewis x epitope recognized by AG107, we performed flow cytometry with neuraminidase-treated ECV304 cells and the AG107 mAb. AG107 binding was only observed with ECV304 cells that had been transfected with GlcNAc6ST-1 or with GlcNAc6ST-1 and Fuc-T VII. It was not observed with parental ECV304 cells or with ECV304 cells transfected with Fuc-T VII alone (Figure 4A and data not shown). To determine if the AG107 epitope generated by GlcNAc6ST-1 was present on CD44, we immunoprecipitated CD44 from parental and GlcNAc6ST-1 and Fuc-T VII transfected ECV304 cells. The AG107 determinant was revealed only after neuraminidase treatment and was present only on GlcNAc6ST-1 expressing ECV304 cells (Figure 4B). Although we cannot exclude the possibility that GlcNAc6ST-4 contributes to a low level of CD44 carbohydrate sulfation, these results implicate GlcNAc6ST-1 in the synthesis of the 6-sulfo LacNAc/Lewis x epitope detected by mAb AG107 on CD44. In addition, PNGase F treatment of immunoprecipitated CD44 decreased the molecular mass of CD44 and reduced the binding of mAb AG107 to CD44 by 82 ± 17% (n = 3). This indicated that the sulfated determinant recognized by AG107 is present largely on N-linked glycans of CD44.
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Discussion |
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In this study, GlcNAc6ST-1 was implicated in the generation of the AG107 epitope, 6-sulfo LacNAc/Lewis x. This epitope was masked by sialic acid and up-regulated on human PBM after TNF-
stimulation and was present on the N-linked glycans of CD44. The factors that make a glycoprotein a good substrate for a particular carbohydrate sulfotransferase are not well understood. In vitro, studies have revealed that human GlcNAc6ST-1, -2, and -4 prefer to sulfate terminal GlcNAc residues (Bowman et al., 2001) and that GlcNAc6ST-1 and -4 show a preference for N-linked glycans (Hiraoka et al., 1999; Uchimura et al., 2000; Uchimura et al., 2002; De Graffenried and Bertozzi, 2003). In Chinese Hamster Ovary cells (CHO), GlcNAc6ST-1 preferred N-linked glycans and was predominantly localized to the trans-Golgi network whereas GlcNAc6ST-2 preferred O-linked glycans and was present throughout the Golgi (De Graffenried and Bertozzi, 2003). Thus, in addition to intrinsic substrate specificity, the expression of the sulfotransferase and its location within the Golgi may also influence which glycoproteins and what glycans are sulfated. Although sialyl 6-sulfo Lewis x present on core 1 and core 2 O-linked glycans contributes to the generation of L-selectin ligands on HEV (Yeh et al., 2001; Hiraoka et al., 2004), it may not function as an L-selectin ligand when present on N-linked glycans (Hiraoka et al., 1999). Preliminary evidence suggests that the induction of the AG107 epitope on CD44 in response to TNF- does not enhance L-selectin-mediated rolling (S.L. Tjew and P. Johnson, unpublished). It had previously been noted that two mAbs that recognize L-selectin ligands, MECA-79 mAb that recognizes 6-sulfo LacNAc on extended core 1 O-linked glycans and the G72 mAb that recognizes sialyl 2, 3 linked 6-sulfo LacNAc/Lewis x structures did not bind to TNF--stimulated SR91 cells (Delcommenne et al., 2002). This further suggests that the epitope on CD44 may be distinct from those present on L-selectin ligands. It is unclear whether the sialylated 6-sulfo LacNAc/Lewis x on N-linked glycans is poorly recognized by G72, or whether the sialic acid linkage to the AG107 epitope is different or polysialylated or indeed, whether the AG107 epitope is masked by neighboring sialic acid residues. Both the precise structure and the function of this sulfated epitope on CD44 on human PBM remain to be established.
Here, we found GlcNAc6ST-1 and -4 transcripts present in human PBM and induced upon exposure to TNF-. Interestingly, GlcNAc6ST-1 and Fuc-T VII transcripts were increased in human umbilical vein endothelial cells treated with interleukin-1ß (IL-1ß), supporting a possible role for GlcNAc6ST-1 and sialyl 6-sulfo Lewis x epitopes in the inflammatory process (Kimura et al., 1999). It is becoming apparent that in chronic inflammatory diseases and in organ rejection, HEV-like structures expressing L-selectin ligands are induced (Rosen, 1999). Initial evidence indicates that the HEV-specific sulfotransferase, GlcNAc6ST-2, is induced under these conditions (Bistrup et al., 2004), but the expression of GlcNAc6ST-1 was not determined.
The AG107 antibody can recognize both desialylated 6-sulfo LacNAc and 6-sulfo Lewis x epitopes (Izawa et al., 2000). By using another mAb, DD2, a 6-sulfo LacNAc-containing epitope has been detected on a subset of human dendritic cells that produce a lot of TNF- in response to the bacterial endotoxin, lipopolysaccharide (Schakel et al., 2002). The DD2 epitope, 6-sulfo LacNAc, was present on the P-selectin glycoprotein ligand-1, PSGL-1, but its function is not known. PSGL-1 can also be modified by a natural killer cell (NK)-restricted keratan sulfate-related lactosamine carbohydrate, which creates a unique binding site for L-selectin (Andre et al., 2000). Originally, TNF--induced sulfation of CD44 on SR91 cells and human monocytes was correlated with induced CD44 binding to hyaluronan (Maiti et al., 1998; Brown et al., 2001). However, subsequent studies in SR91 cells revealed that the sulfation of CD44 was complex, occurring on chondroitin sulfate as well as on both N- and O-linked glycans (Delcommenne et al., 2002). Preliminary data suggest that there is not always a correlation between the expression of the AG107 epitope and hyaluronan binding and so the function of this epitope remains to be established (S.L. Tjew and P. Johnson, unpublished). Overall, the induction of 6-sulfo LacNAc/Lewis x on human monocytes in response to TNF- suggests a potential role in the inflammatory process. Interestingly, an increase in the sulfation of high molecular weight glycoconjugates occurs in the lungs of cystic fibrosis patients (Cheng et al., 1989) and exposure of human bronchial mucosa to TNF- results in increased activity of several glycosyl- and sulfo-transferases, including both galactose 3-O- and GlcNAc 6-O-sulfotransferase activities (Delmotte et al., 2002). This suggests that inflammatory conditions can influence the sulfation state of glycoconjugates. Here, inflammatory conditions induce the expression of 6-sulfo LacNAc/Lewis x on activated human monocytes, which may then contribute to the inflammatory process or towards innate immunity.
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Materials and methods |
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Reagents and Abs
The rat anti-mouse/human CD44 mAb, IM7.8.1 (TIB-235), mouse anti-human CD44 mAb Hermes-3 (HB-9480), available from American Type Culture Collection (Manassas, VA) and mouse IgM mAb, AG107, anti-6-sulfo LacNAc/Lewis x (Izawa et al., 2000
) were used. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM and goat anti-rat Abs were purchased from Jackson ImmunoResearch Laboratories (Mississauga, Ontario). Phycoerythrin (PE)-conjugated mouse anti-human CD14 was from Caltag Laboratories (Burlingame, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-mouse IgM were from Southern Biotechnology Associates (Birmingham, AL).
Cells and cell lines
Human PBMC were isolated and cultured in RPMI 1640 with 10% fetal calf serum (FCS) and stimulated with 20 ng/mL recombinant human TNF- (rhTNF-; R & D Systems, Minneapolis, MN) for 72 h. CD14 was used as a marker for human PBM, and CD14+ PBMC were positively selected with anti-CD14-conjugated magnetic beads (M450; Dynal, Lake Success, NY) as described previously (Brown et al., 2001). CD14– PBMC contained 1% or less CD14+ cells. The human myelo-leukemic cell line, SR91 (Klingemann et al., 1994), was cultured in RPMI/10% FCS and stimulated with 10 ng/mL rhTNF- for 24 h. ECV304, ECV304 transfected with GlcNAc6ST-1, Fuc-T VII, or both (Kimura et al., 1999) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) /10% FCS, with 6 U/mL hygromycin B (Calbiochem, La Jolla, CA) for GlcNAc6ST-1 transfectants, or 100 mg/mL active Geneticin (Invitrogen, Burlington, Ontario) for Fuc-T VII transfectants.
Neuraminidase treatment and flow cytometry
Cells were resuspended at 1 x 107 cells/mL and incubated with 0.05 U/mL neuraminidase (from Vibrio cholerae, Roche Diagnostics, Laval, Quebec) for 1 h to remove terminal sialic residues and flow cytometry was performed with IM7.8.1 and AG107 mAbs, as described previously (Brown et al., 2001; Delcommenne et al., 2002).
Immunoprecipitation, PNGase F digestion, and western blot analysis
CD14+ PBMC, ECV304, and SR91 cells were lysed in 10 mM Tris, pH 7.2, containing 140 mM KCl, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 1 µg/mL pepstatin. CD44 was immunoprecipitated with IM7-conjugated Sepharose beads for 2 h at 4°C and then treated overnight with or without 500 U PNGase F (New England Biolabs, Mississauga, Ontario) in 20 µL 50 mM sodium phosphate buffer, pH 7.5, containing 0.5% sodium dodecyl sulfate (SDS), 1% NP-40, and 1% ß-mercaptoethanol. To stop the reaction, 800 µL acetone was added and incubated at –20°C for 1 h. The samples were centrifuged, resuspended in reducing sample buffer, separated by 7.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to polyvinylidene difluoride (PVDF) membrane. The dried membranes were incubated with mAb AG107 hybridoma supernatant, as described in Delcommenne et al. (2002). Membranes were stripped by incubating with 100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.7 for 90 min at 70°C, dried, then incubated for 1 h with 1/50 Hermes-3 hybridoma supernatant followed by 1/5000 HRP-conjugated goat anti-mouse IgG, and detected with ECL (Amersham Biosciences). Western blots were converted to digital images and bands quantified by using the VersaDoc imaging system (Bio-Rad, Mississauga, Ontario).
[35S]Sulfate labeling
ECV304 cells were seeded onto 60 mm plates in the presence or absence of 100 µCi/mL Na235SO4 (ICN Biochemicals, St. Laurent, Quebec) such that they were 80% confluent the next day. CD44 from ECV304 cells was immunoprecipitated, subjected to SDS–PAGE and transferred to PVDF membrane, and exposed to film with an intensifying screen at –80°C for 24 h before western blotting with the mAb AG107.
Semiquantitative RT–PCR
The following primers were used to amplify each of the following human sulfotransferase cDNAs. The melting temperature used during polymerase chain reaction (PCR) and fragment size are as indicated: for GlcNAc6ST-1 (5'-GAGGTGTTCTTTCTCTACGAGCC-3' and 5'-CCACGAAAGGCTTGGAGGAGG-3'), 51°C, 847 bp (Li and Tedder, 1999); for GlcNAc6ST-2 (5'-TGACATCATCCCACAAGATGAAATCATCCC-3' and 5'-GATTTGCTCAGGGACAGTCCAGGT-3'), 53°C, 742 bp; for GlcNAc6ST-3 (5'-ACCATCAGCAAGCAGGACGTAT-3' and 5'-TCTAAGGCCCAGAGTTCTCAGTCA-3'), 53°C, 844 bp; for GlcNAc6ST-4 (5'-GAACCAGTCTCCTCGGTTCCCAAG-3' and 5'-ACCGGTCAGGAAGAAATCGGCGCG-3'), 51–55°C, 793 bp; for GlcNAc6ST-5 (5'-CGTGTTTGATGCCTATCTGCCTTG-3' and 5'-TGCGAGGCGGTGGATGAT-3'), 51°C, 851 bp; for ß-actin (5'-GACTACCTCATGAAGATCCT-3' and 5'-ATCCACATCTGCTGGAAGGT-3'), 49–55°C, 512 bp; for C6ST-1 (5'-AAGGGTCTCAGACAAGCTGAAGCA-3' and 5'-CACTTCTTCCAGGTCTTATACTTGCCG-3'), 49°C, 834 bp; and for KSGal6ST (5'-TTCACCGCCAAGTCCTTTCACA-3' and 5'-CCGTAGATCTCCTCGGTCTTCTTCAT-3'), 49°C, 877 bp. Briefly, 5 µg of total RNA isolated from 5 x 106 SR91 cells or 0.6–2.5 µg RNA from 1 x 106 positively selected CD14+ PBMC by using RNeasy Mini kit (Qiagen, Chatsworth, CA) was reverse transcribed with Superscript II (Invitrogen), according to manufacturers’ instructions and an aliquot subjected to PCR with Platinum Pfx polymerase (Invitrogen) in 50 µL under the following conditions: 1 cycle at 94°C for 5 min; 30–35 cycles at 94°C for 30 s, 49–55°C for 30 s, and 68°C for 75 s; 1 cycle at 68°C for 10 min. Transcripts were monitored over several cycles and determined to be within the linear range when amplified for 23 cycles for ß-actin, 30 cycles for GlcNAcST from SR91 cells, and 32–35 cycles for CD14+ PBMC. Twenty microliters of the PCR product was electrophoresed in 1% agarose gel, stained with ethidium bromide and visualized under ultraviolet light. Sulfotransferase transcripts were normalized to ß-actin transcripts generated in the same PCR tube.
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Acknowledgements |
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We thank all the volunteers who donated blood. This work was supported by grants from the Canadian Institutes for Health Research (CIHR) and the British Columbia and Yukon Heart and Stroke Foundation of Canada (BC & Y HSF). S.T. is supported by a research traineeship from the Natural Sciences and Engineering Research Council of Canada. K.B. was supported by research traineeships from BC & Y HSF and the Michael Smith Foundation for Health Research. P. J. is a scientist of the CIHR.
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Abbreviations |
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FCS, fetal calf serum; Fuc-T VII,
1,3 fucosyltransferase VII; Gal, Galactosamine; GlcNAc6ST, N-acetylglucosamine 6-O-sulfotransferase (see [Fukuda et al., 2001] for the definition of sulfotransferase nomenclature); HEV, high-walled endothelial venules; LacNAc, N-acetyllactosamine; mAb, monoclonal antibody; PBM, peripheral blood monocytes; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; RT–PCR, reverse transcriptase polymerase chain reaction; TNF-, tumor necrosis factor alpha. |
References |
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