Glycobiology Advance Access originally published online on August 3, 2005
Glycobiology 2005 15(12):1349-1358; doi:10.1093/glycob/cwj024
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Differential expression and enzymatic properties of GalNAc-4-sulfotransferase-1 and GalNAc-4-sulfotransferase-2
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110
1 To whom correspondence should be addressed; e-mail: baenziger{at}pathology.wustl.edu
Received on May 30, 2005; revised on July 8, 2005; accepted on July 22, 2005
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Abstract |
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We have cloned two GalNAc-4-sulfotransferases, GalNAc-4-ST1 and GalNAc-4-ST2, that transfer sulfate to terminal ß1,4-linked GalNAc. In conjunction with the action of protein-specific ß1,4GalNAc-transferases, GalNAc-4-ST1 and GalNAc-4-ST2 account for the presence of terminal ß1,4-linked GalNAc-4-SO4 on glycoproteins such as lutropin, thyrotropin (TSH), proopiomelanocortin (POMC), carbonic anhydratase-VI (CA-VI), and tenascin-R. GalNAc-4-ST1 and GalNAc-4-ST2 can be distinguished by their differing specificity for oligosaccharide acceptors and temperature lability. The differences in properties have been used to show that the levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity are proportionate to the levels of their respective transcripts. Furthermore, we have found that both transcript and activity levels of GalNAc-4-ST1 and GalNAc-4-ST2 vary widely among different tissues indicating that the regulation of their expression differs. Differences in specificity and the regulation of expression may account for existence of two GalNAc-4-sulfotransferases in vivo. The highest levels of both GalNAc-4-ST1 and GalNAc-4-ST2 transcripts are present in the pituitary of the mouse with multiple cell types that produce glycoproteins terminating with GalNAc-4-SO4. Genetic ablation of both GalNAc-4-ST1 and GalNAc-4-ST2 may be necessary to alter the pattern and/or extent of sulfate addition to terminal ß1,4GalNAc in tissues such as pituitary.
Key words: GalNAc-4-sulfotransferase/ogliosaccharides/specificity/sulfate/transcript levels
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Introduction |
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Many glycoproteins bear N- or O-linked oligosaccharides that terminate with ß1,4-linked GalNAc-4-SO4. These include the glycoprotein hormones lutropin (LH) and thyrotropin (TSH) (Baenziger and Green, 1988
; Green and Baenziger, 1988a,b), proopiomelanocortin (POMC) (Skelton et al., 1992; Siciliano et al., 1993, 1994), carbonic anhydratase-VI (CA-VI) (Hooper et al., 1995), tenascin-R (Woodworth et al., 2001), sialoadhesin, and CD45 (Martinez-Pomares et al., 1999). Protein-specific ß1,4GalNAc-transferase and GalNAc-4-sulfotransferase activities that act sequentially to generate these structures are present in many tissues including those known to synthesize glycoproteins bearing terminal GalNAc-4-SO4 (Dharmesh et al., 1993). We and others have cloned two cDNAs, designated GalNAc-4-ST1 (Okuda et al., 2000a, 2003; Xia et al., 2000; Hiraoka et al., 2001) and GalNAc-4-ST2 (Hiraoka et al., 2001; Kang et al., 2001; Okuda et al., 2003), from human and mouse cDNA libraries that transfer sulfate to terminal ß1,4-linked GalNAc in vitro. GalNAc-4-ST1 and GalNAc-4-ST2 are closely related with 47% identity in their complete amino acid sequence and 67% identity within their catalytic domains. GalNAc-4-ST1 and GalNAc-4-ST2 are members of a family of sulfotransferases that includes HNK-1 sulfotransferase (Bakker et al., 1997; Ong et al., 1998), chondroitin-4-sulfotransferase-1 (Hiraoka et al., 2000; Okuda et al., 2000b; Yamauchi et al., 2000), chondroitin-4-sulfotransferase-2 (Hiraoka et al., 2000), chondroitin-4-sulfotransferase-3 (Kang et al., 2002), and dermatan-4-sulfotransferase-1 (Evers et al., 2001). Northern blot analyses indicate that the GalNAc-4-sulfotransferases are expressed in a number of tissues, with high levels in the pituitary and brain. The existence of two GalNAc-4-sulfotransferases suggests that there are differences in their specificity, properties, and/or the regulation of their expression. We have used quantitative polymerase chain reaction (PCR) to determine the steady state levels of GalNAc-4-ST1 and GalNAc-4-ST2 transcripts in various tissues. In addition, we have examined the specificity and properties of recombinant forms GalNAc-4-ST1 and GalNAc-4-ST2. Differences in their temperature lability have allowed us to establish that the levels of GalNAc-4-ST1 and GalNAc-4-ST2 transcripts reflect the relative levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity in tissues. Both the relative and absolute levels of GalNAc-4-ST1 and GalNAc-4-ST2 differ markedly among tissues.
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Results |
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GalNAc-4-ST1 and GalNAc-4-ST2 transcript levels in adult mice
Transcript levels for GalNAc-4-ST1 and GalNAc-4-ST2 were determined using real-time PCR as shown in Figure 1A. GalNAc-4-ST1 and GalNAc-4-ST2 transcript levels are highest in the pituitary where glycoproteins known to bear terminal ß1,4-linked GalNAc-4-SO4 are known to be synthesized including LH, TSH, and POMC (Baenziger and Green, 1991
; Skelton et al., 1992). Transcripts for GalNAc-4-ST1 and/or GalNAc-4-ST2 are present in many tissues that we have previously determined either express GalNAc-4-sulfotransferase activity (Dharmesh et al., 1993). The same tissues can be stained for the presence of glycoproteins terminating with ß1,4-linked GalNAc-4-SO4 using Cys-Fc, a chimeric protein containing the Cysteine-rich domain of the Mannose/GalNAc-4-SO4-specific receptor that is highly specific for this structure (Fiete et al., 1998; Woodworth et al., 2001).
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The ratio of GalNAc-4-ST1 : GalNAc-4-ST2 transcripts was determined (Figure 1B) and revealed that GalNAc-4-ST1 transcripts dominated in multiple regions of the brain as well as in kidney, ovary, and uterus. No example of a tissue containing exclusively GalNAc-4-ST1 transcript was found. This was distinct from the situation with GalNAc-4-ST2 where multiple tissues appeared to express almost exclusively GalNAc-4-ST2 transcripts as evidenced by the high GalNAc-4-ST2 : GalNAc-4-ST1 ratios seen in heart, spleen, testis, liver, and salivary gland (Figure 1C). The different transcript levels seen for GalNAc-4-ST1 and GalNAc-4-ST2 in tissues as well as the different ratios of GalNAc-4-ST1 and GalNAc-4-ST2 transcript levels in tissues indicate that the expression of GalNAc-4-ST1 and GalNAc-4-ST2 are not regulated in the same manner. These differences raise the possibility that the specificity and/or properties of these sulfotransferases are not the same.
Kinetic properties of GalNAc-4-ST1 and GalNAc-4-ST2
We examined the properties of recombinant GalNAc-4-ST1 and GalNAc-4-ST2 expressed in Chinese hamster ovary (CHO) cells. The apparent Km for SO4 addition to the disaccharide acceptor GalNAcß1,4GlcNAcß-(CH2)8-COOCH3 (GGn-MCO) is 8.8 µM for GalNAc-4-ST1 and 11.1 µM for GalNAc-4-ST2 (Figure 2A and B). Furthermore, the Vmax per microgram of cell extract is similar for the two sulfotransferases. GalNAc-4-ST1 and GalNAc-4-ST2 have similar apparent Km for 3'-phosphoadenosine-5'-phosphosulfonate (PAPS) of 2.31 µM and 1.96 µM, respectively (Figure 2C and D).
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Specificity of GalNAc-4-ST1 and GalNAc-4-ST2 for oligosaccharides terminating with ß1, 4-linked GalNAc
The oligosaccharides shown in Table I were compared as substrates for GalNAc-4-ST1 and GalNAc-4-ST2. The levels of expression were similar based on western blot analysis using an antibody directed at the V5 epitope tag (not shown). A catalytic efficiency (Vmax/Km) was calculated for each substrate to allow comparison GalNAc-4-ST1 and GalNAc-4-ST2. GalNAc-4-ST1 and GalNAc-4-ST2 differ in their efficiencies of sulfate transfer to the oligosaccharides tested. Both sulfotransferases display a reduced rate of sulfate transfer when the ß-linked mannose bears substituents arising from both C3 and C6 (compare compounds 3 and 4 with compounds 1, 2, and 5 in Table I). The reduced catalytic efficiency is not overcome by the presence of a second terminal ß1,4-linked GalNAc acceptor on compound 4 but reduced even further. In contrast, the presence of two branches arising from C2 and C6 of the 
1,6-linked Man and terminating with GalNAc in compound 6 enhances transfer by GalNAc-4-ST2 and does not reduce the catalytic efficiency for GalNAc-4-ST1 significantly. Furthermore, GalNAc-4-ST2 transfers sulfate more efficiently to the O-linked structure, compound 7, than does GalNAc-4-ST1. The properties of GalNAc-4-ST1 and GalNAc-4-ST2 suggest that GalNAc-4-ST2 is more highly active with tri- and tetra-branched N-linked structures and with O-linked structures than is GalNAc-4-ST1.
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Cation and pH dependence
GalNAc-4-ST1 and GalNAc-4-ST2 both demonstrate a preference for Mg2+ over either Mn2+ or Ca2+ (Figure 3). In the presence of 1 mM ethylene diamine tetraacetic acid (EDTA) and no added divalent cation GalNAc-4-ST2 has little activity whereas GalNAc-4-ST1 retains as much as 60% of the maximal activity obtained with 5 mM Mg2+. GalNAc-4-ST2 displays a clear concentration dependence on Mg2+ over a wider range of concentrations than GalNAc-4-ST1. Both sulfotransferases have identical pH optima of 7.6 using imidazole as the buffering agent (Figure 4).
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Temperature lability and tissue distribution
We previously noted that it was necessary to perform GalNAc-4-sulfotransferase assays at 28°C because of the rapid loss of activity at 37°C when determining enzyme levels following solublization from tissue with Triton X-100 (Skelton et al., 1991). Recombinant GalNAc-4-ST1 that is solublized with either 2% Triton X-100 (not shown) or T-PER is stable to incubation at 37°C in either the presence or absence 15% glycerol. Recombinant GalNAc-4-ST2 solublized with 2% Triton X-100 in 20 mM Tris, 200 mM NaCl is labile to incubation at 37°C in either the presence or absence of glycerol (not shown). In contrast, GalNAc-4-ST2 solublized in T-PER is stable to incubation at 37°C if 15% glycerol is present (Figure 5B) but is labile if glycerol is not present (Figure 5D). At 37°C recombinant GalNAc-4-ST2 has a t1/2 of 3.6 min. Recombinant human GalNAc-4-ST1 and GalNAc-4-ST2 displayed a similar difference in temperature lability (not shown). When recombinant GalNAc-4-ST1 and GalNAc-4-ST2 were combined in different proportions the amount of activity remaining following incubation at 37°C was equal to the proportion of GalNAc-4-ST1 in the sample (not shown).
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We took advantage of the difference in temperature lability of GalNAc-4-ST1 and GalNAc-4-ST2 at 37°C to determine what proportion of the GalNAc-4-sulfotransferase activity reflects GalNAc-4-ST1 as opposed to GalNAc-4-ST2 in various tissues as shown in Figure 6. Total GalNAc-4-sulfotransferase activity varied widely among the tissues tested with salivary gland extracts having the highest levels per microgram of protein extract followed by pituitary, cerebellum, ovary, uterus, kidney medulla and testis (Figure 6A). The activity in salivary gland and testis that have ratios of GalNAc-4-ST2 : GalNAc-4-ST1 transcript
100 (Figure 1C) is nearly all labile to incubation at 37°C indicating that GalNAc-4-ST2 accounts for virtually all of the activity in these tissues. The kinetics for loss of activity at 37°C in salivary was identical to that seen with recombinant GalNAc-4-ST2 in the absence of glycerol (not shown). In contrast tissues that have GalNAc-4-ST1 : GalNAc-4-ST2 transcript ratios of >2.5 display little lability at 37°C (Figures 1B and 6B). Extracts of pituitary display a 20% decrease in activity at 37°C consistent with the presence of both activities in the pituitary. GalNAc-4-ST1 and GalNAc-4-ST2 transcript levels are thus reflective of the relative levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity present in cells and tissues.
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Regulation of GalNAc-4-ST1 and GalNAc-4-ST2 transcript and activity levels
We previously reported that the levels of GalNAc-4-sulfotransferase activity in cultured AtT20 and T3 cells can be modulated by incubation in the presence of dimethylsulfoxide (DMSO) and retinoic acid, respectively (Skelton, 1993). The predominant transcript in both cell lines is GalNAc-4-ST1; however, relative to 18s rRNA the steady state GalNAc-4-ST1 transcript level is 88-fold greater in T3 cells than AtT20 cells (Figure 7). The GalNAc-4-sulfotransferase activity in cell extracts and medium from AtT20 and T3 cells is not labile to incubation at 37°C (not shown) indicating that the vast majority of sulfotransferase activity arises from GalNAc-4-ST1. Both cell lines release GalNAc-4-sulfotransferase activity into the medium (not shown). The levels of GalNAc-4-sulfotransferase activity are 3- to 5-fold higher in T3 cells than AtT20 cells (Table II). Treatment with DMSO increases both the GalNAc-4-ST1 transcript and activity levels in AtT20 cells. Treatment with retinoic acid increases GalNAc-4-ST1 activity only modestly in either T3 cell extracts or medium (not shown) even though GalNAc-4-ST1 transcript levels are increased. Thus, it appears that even though treatment with retinoic acid can increase the levels of GalNAc-4-ST1 transcript in T3 cells, the cells are already producing maximal levels of GalNAc-4-ST1. In contrast, the GalNAc-4-ST1 transcript levels are low in AtT20 cells compared to T3 cells and the increase in GalNAc-4-ST1 transcripts induced by DMSO is accompanied by a 1.7- to 1.9-fold increase in the amount of GalNAc-4-ST1 activity in the cells and medium (not shown).
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Discussion |
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The existence of two closely related GalNAc-4-sulfotransferases that both transfer sulfate to terminal ß1,4-linked GalNAc suggests that they may differ in their properties or expression and, as a consequence, their function in vivo. When transfected into cells that produce glycoproteins with N-linked oligosaccharides terminating with ß1,4-linked GalNAc, GalNAc-4-ST1 and GalNAc-4-ST2 are both able to quantitatively modify terminal GalNAc moieties with SO4, indicating that these GalNAc-4-sulfotransferases are highly active in vivo as well as in vitro. GalNAc-4-ST1 and GalNAc-4-ST2 transcripts are expressed in numerous tissues in the mouse; however, both the steady state levels of these transcripts and their levels relative to each other vary widely in tissues. The steady state levels of transcript for both GalNAc-4-ST1 and GalNAc-4-ST2 are highest in the pituitary (Figure 1A) where glycoproteins bearing terminal GalNAc-4-SO4 are synthesized by gonadotrophs, thyrotrophs and corticotrophs. High levels of one or both transcripts are also found in the medulla of the kidney, spleen, ovary, uterus, salivary gland and cerebellum.
Differences in the relative abundance of GalNAc-4-ST1 and GalNAc-4-ST2 transcripts can be more readily appreciated from the ratios of GalNAc-4-ST1 : GalNAc-4-ST2 and GalNAc-4-ST2 : GalNAc-4-ST1 as shown in Figure 1B and C, respectively. Tissue such as heart, spleen, testis, liver and salivary gland express predominantly GalNAc-4-ST2 and little, if any, GalNAc-4-ST1. In contrast, pituitary, multiple regions of the brain, ovary and kidney medulla express higher levels of GalNAc-4-ST1 than GalNAc-4-ST2. Each of the tissues expressing GalNAc-4-ST1 transcript also had significant levels of GalNAc-4-ST2 transcript (Figure 1A). The steady state the transcript levels we have determined for GalNAc-4-ST1 and GalNAc-4-ST2 by quantative reverse transcriptase PCR (RT–PCR) using SYBR Green are in agreement with both the catalytic properties and temperature lability of the GalNAc-4-sulfotransferase activities is the tissues that were examined. Our conclusions differ from those of Okuda et al. (2003) who used a "semiquantitative" form of RT–PCR analysis and reported that brain and kidney contained similar levels of GalNAc-4-ST1 and GalNAc-4-ST2 transcripts whereas pituitary contained almost exclusively GalNAc-4-ST2 transcript.
The temperature lability of the GalNAc-4-sulfotransferase activity in extracts prepared from salivary gland and testis (Figure 6B) indicates that GalNAc-4-ST2 is the predominant if not exclusive activity present in these tissues. Furthermore, the highest level of GalNAc-4-ST2 transcript and GalNAc-4-sulfotransferase activity are found in the salivary gland. In contrast, the GalNAc-4-sulfotransferase activity in cerebellum, ovary and kidney medulla demonstrates little or no temperature lability in agreement with the predominance of GalNAc-4-ST1 transcripts in these tissues. This was further confirmed by examining the kinetic properties of the GalNAc-4-sulfotransferases in cerebellum and salivary gland extracts using substrate 3 in Table I. The apparent Km for sulfate transfer was 77.9 µM for cerebellum and 8.4 µM for salivary gland in agreement with the apparent Km obtained for recombinant GalNAc-4-ST1 and GalNAc-4-ST2 of 70.8 µM and 14.7 µM, respectively. Thus, GalNAc-4-ST1 and GalNAc-4-ST2 transcript levels reflect the relative levels of GalNAc-4-sulfotransferase 1 and GalNAc-4-sulfotransferase 2 activity, respectively, in these tissues.
Based on both the lack of temperature lability and the greater steady state level of GalNAc-4-ST1 relative to GalNAc-4-ST2 transcript in AtT20 and T3 cells, GalNAc-4-ST1 is the predominant sulfotransferase expressed in these murine cells. T3 cells have 88-fold higher GalNAc-4-ST1 transcript levels than AtT20 cells yet only produce 4-fold more GalNAc-4-ST1 enzymatic activity. Following treatment with DMSO or retinoic acid there are 27- and 16-fold increases in GalNAc-4-ST1 transcript in AtT20 and T3 cells, respectively, but the amount of intracellular activity only increases by 1.9- and 1.2-fold. The increase in activity is thus not proportionate to the increase in transcript in these cells. GalNAc-4-ST2 transcript levels also increase with these treatments; however, relative to GalNAc-4-ST1 the GalNAc-4-ST2 transcript levels remain low. In addition, based on temperature lability GalNAc-4-ST2 activity does not account for a detectable proportion of the total sulfotransferase activity from these cells.
The response of T3 and AtT20 cells to retinoic acid and DMSO supports the conclusion that the expression of GalNAc-4-ST1 and GalNAc-4-ST2 are differentially regulated in cells and tissues. As a result either GalNAc-4-ST1 or GalNAc-4-ST2 may predominate or both may be present in a given cell type or tissue. We are currently exploring in what manner the expression of these sulfotransferases changes during events such as development and the ovulatory cycle.
GalNAc-4-ST1 and GalNAc-4-ST2 differ in their specificity for oligosaccharide acceptors. With the exception of compound 1 in Table I, GalNAc-4-ST2 has a catalytic efficiency that is 3.5- to 6.0-fold higher for oligosaccharides acceptors containing ß1,4-linked GalNAc than that obtained with GalNAc-4-ST1. This largely reflects the lower apparent Km seen for GalNAc-4-ST2 as compared to GalNAc-4-ST1. The presence of a second branch arising from the ß-linked Man reduces the catalytic efficiency of sulfate transfer for both sulfotransferases. This effect is not overcome by the presence of a second terminus with ß1,4-linked GalNAc; i.e., compound 4. In contrast, the presence of a second branch terminating with ß1,4-linked GalNAc has little effect of the catalytic efficiency of GalNAc-4-ST1 when arising from the 1,6-linked GlcNAc (compare compound 5 versus 6 in Table I) whereas the catalytic efficiency increases 2.3-fold for GalNAc-4-ST2. In addition, GalNAc-4-ST2 has a 5.3-fold higher catalytic efficiency for transfer of sulfate to the O-linked type structure, compound 7 in Table I, than does GalNAc-4-ST1. This suggests that GalNAc-4-ST2 may be more effective than GalNAc-4-ST1 for modifying O-linked structures such as are found on mucins and POMC (Siciliano et al., 1994) and the more highly branched complex N-linked structures we found on CA-VI from submaxillary glands (Hooper et al., 1995). The higher catalytic efficiency with O-linked and more highly branched N-linked structures may explain the predominance of GalNAc-4-ST2 in salivary glands that produce such structures.
GalNAc-4-ST1 and GalNAc-4-ST2, although closely related structurally, display significant differences in specificity and expression. GalNAc-4-ST1 and GalNAc-4-ST2 are capable of adding sulfate to the same spectrum of oligosaccharides; however, their efficiency and the hierarchy of structures that they modify differ. The presence of both GalNAc-4-sulfotransferases in tissues such as pituitary and brain may reflect the amount of GalNAc-4-SO4-bearing structures that are synthesized and the importance of modifying virtually all terminal ß1,4-linked GalNAc moieties with sulfate. The marked differences in the expression of GalNAc-4-ST1 and GalNAc-4-ST2 in tissues suggests they have roles related to the differences in their specificity. Further definition of the full spectrum of glycoproteins that bear oligosaccharides terminating with ß1,4-linked GalNAc-4-SO4 as well as genetically ablated animals will help to assess the function of these sulfotransferases and the unique sulfated structures they produce.
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Materials and methods |
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Preparation of GalNAc-4-sulfotransferase from cultured cells and tissues
cDNAs encoding the mouse orthologues of human GalNAc-4-ST1 and GalNAc-4-ST2 were cloned from a mouse pituitary library by PCR and ligated into the expression vector pcDNA3.1 and pcDNA3.1-V5His (Invitrogen, Carlsbad, CA). The cloned cDNAs were found to be identical to AB106878 [GenBank] and AB106879 [GenBank] (Okuda et al., 2003
), respectively, and were designated pcDNA3.1-mGalNAc-4-ST1, pcDNA3.1-mGalNAc-4-ST2, pcDNA3.1-mGalNAc-4-ST1-V5His and pcDNA3.1-mGalNAc-4-ST2-V5His. Forms of mGalNAc-4-ST1 and mGalNAc-4-ST2 that were epitope tagged at their C-termini with the epitope V5 and were fully active.
CHO cells, which do not express endogenous GalNAc-4-sulfotransferase activity when tested with GGnM-MCO (Xia et al., 2000), were cultured in 100-mm plates and transfected with pcDNA3.1-mGalNAc-4-ST1, pcDNA3.1-mGalNAc-4-ST2, pcDNA3.1-mGalNAc-4-ST1-V5His or pcDNA3.1-mGalNAc-4-ST2-V5His cDNA using Lipofectamine (Invitrogen) (13 µg DNA : 35 µg Lipofectamine). Six hours after transfection, 20% fetal bovine serum in OptiMEM (Gibco/BRL) was added to the cells, and they were cultured for an additional 48 h. The transfected CHO cells were washed with phosphate-buffered saline (PBS) and lyzed by adding 250 µL of T-PER tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL) containing protease inhibitor (Complete, Boehringer Mannheim GmbH, Mannheim, Germany) and incubating for 20 min at 4°C with periodic vortexing after scraping the cells off the plate. The extract was sedimented at 12,000 rpm in a microcentrifuge for 15 min at 4°C, the supernatant was removed and brought to 15% glycerol (vol : vol) before storing at –80°C. When utilized to examine differences in temperature lability equal aliquots of extract were prepared without the addition of glycerol before storage.
Tissues were harvested from mice, immediately frozen in liquid nitrogen, and stored at –80°C. Frozen tissues were placed in ice cold T-PER buffer (1:1 wt : vol) and disrupted using a 1-mL ground glass homogenizer (Kimble Kontes, Vineland, NJ) on ice. The homogenates were centrifuged at 12,000 rpm in a microcentrifuge for 15 min at 4°C. The supernatants were stored at –80°C in single use aliquots in either the presence or absence 15% glycerol.
Synthesis of oligosaccharide acceptors
2-Acetamido-2-deoxy-ß-D-glucopyranosyl-(CH2)8COOCH3 (Gn-MCO) was purchased from Rose Scientific (Edmunton, Alberta, Canada). A mutant form of recombinant bovine ß1,4-galactosyltransferase GalT1(Y289L) produced in Escherichia coli that transfers GalNAc to terminal GlcNAc was generously provided by Dr. B. Ramakrishnan and Dr. P.K. Qasba (NCI-Frederick, NIH, Frederick, MD) (Ramakrishnan and Qasba, 2002) and used to synthesize GGn-MCO. The synthetic reaction contained 3.75 mM Gn-MCO, 3.75 mM UDP GalNAc, 2.8 mM phosphoenolpyruvate, 4.2 mM ß-NADH, 3.5 unit/mL L-lactate dehydrogenase, 70 unit/mL pyruvate kinase, 100 mM cacodylate pH 7.4, 5 mM MnCl2, 250 mM KCl, and 20 µg/mL GalT1(Y289L). Following incubation at 37°C for 24 h, >95% of the Gn-MCO was converted to GGn-MCO as assessed by high performance liquid chromatography on a MicroPak AX-5 column (Varian Associates, Sunnyvale, CA) using a gradient of acetonitrile : water 80:20–40:60 over 100 min. The GGn-MCO was isolated by loading the enzyme reaction onto a Sep-PAK(C18) cartridge (360 mg) (Waters Associates, Milford, MA). After washing the cartridge with 30 mL of water and 10 mL of 8% methanol, the GGn-MCO was eluted with 10 mL of 30% methanol, lyophilized, and dissolved in water. The preparation of the additional acceptors utilized in Table I has been described previously (Hooper et al., 1995). Concentrations were based on monosaccharide analysis.
GalNAc-4-sulfotransferase enzyme assay
The GalNAc-4-sulfotransferase enzyme assay was carried out at 28°C for 2 h as described (Skelton et al., 1991). Each 50-µL reaction contained 15 mM N-[2-hydroxyethyl]piperazine-N"-2-ethanesulfonic acid (HEPES), pH 7.4, 1% Triton X-100, 40 mM 2-mercaptoethanol, 10 mM NaF, 1 mM ATP, 4 mM magnesium acetate, 13% glycerol, protease inhibitors, 250 pmole of unlabeled PAPS, 1 x 106 cpm of [35S] PAPS, and 20 µM GGn-MCO and enzyme. [35S]SO4-4-GGn-MCO was separated from [35S] PAPS and labeled endogenous acceptors by passage over a Sep-Pak C18 cartridge (Waters). Control reactions were carried out in the absence of either substrate or enzyme.
Acceptor substrate specificity
The kinetic properties of GalNAc-4-ST1 and GalNAc-4-ST2 and GalNAc-4-sulfotransferase activities in tissues were examined as described (Skelton et al., 1991; Hooper et al., 1995) using 0–50 µM concentrations of each of the oligosaccharide acceptors shown in Table I and 0––6 µM concentrations of PAPS. Catalytic efficiencies (Vmax/Km) were calculated for each substrate.
Divalent cation and pH dependence
Transferase reactions were made 1 mM with respect to EDTA and sufficient Mg2+, Mn2+ or Ca2+ added to bring the concentration of free divalent cation to 2.5, 5 or 10 mM. The impact of pH was determined using 15 mM imidazole HCl in place of HEPES buffer over a pH range of 6.2–7.8.
Temperature lability
Tissue and cell extracts were incubated at 4° or 37°C in presence and absence of 15% glycerol for 30 min and then placed on ice. The amount of GalNAc-4-sulfotransferase activity remaining was then determined as described above.
Induction of enzymatic activity in AtT20 and T3 cells
AtT20 and T3 cells were incubated in the presence of 1% (vol : vol) DMSO or 10 µM retinoic acid, respectively, to induce increased expression of GalNAc-4-sulfotransferase activity (Skelton et al., 1991). Following incubation for the specified times cells were extracted with T-PER buffer as described above and GalNAc-4-sulfotransferase activity levels and temperature lability determined.
Quantitative PCR
Total RNA was extracted from mouse tissues using Trizol reagent (Invitrogen) and further purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Two microgram of total RNA was used to synthesize the first-strand cDNA with Ominiscript Reverse Transcriptase (Qiagen). Quantitative PCR was performed using an ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) and the following primers for GalNAc-4-ST1, GalNAc-4-ST2 and 18s rRNA: ST1 forward, 5'-CCAGCATGATAGCCACTTGAGGATA-3'; ST1 reverse, 5'-CGCCTGCGCTGCCGGAGGCGCAAA-3'; ST2 forward, 5'-GGAGTATCGGTGACTACGGGAA-3'; ST2 reverse, 5'-GGCTGATCTTCCTTAAAACCCTT-3'; 18s rRNA forward, 5'-CGATGGTAGTCGCCGTGCCTA-3' and 18s rRNA reverse, 5'-CCCTCCAATGGATCCTCGTTAAA-3'.
PCRs were carried out using a QunatiTect SYBR Green PCR Kit (Qiagen) under the following conditions: incubation at 95°C for 10 min, 40 cycles of reaction including denaturation at 95°C for 15 s and extension at 60°C for 30 s. Each sample was tested in triplicate. Slopes for GalNAc-4-ST1 and GalNAc-4-ST2 are parallel. Data for Q-RT–PCR were analyzed using standard curve software (ABI Prism 7000 Sequence Detection System) and were normalized to 18s rRNA.
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Acknowledgements |
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This work was supported by National Institutes of Health Grant R01-DK41738 to J.U.B.
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Abbreviations |
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CHO, Chinese hamster ovary; DMSO, dimethylsulfoxide; EDTA, ethylene diamine tetraacetic acid; GalNAc-4-ST1, GalNAc-4-sulfotransferase-1; GalNAc-4-ST2, GalNAc-4-sulfotransferase-2; GGn-MCO, GalNAcß1,4GlcNAcß-(CH2)8-COOCH3; Gn-MCO, 2-acetamido-2-deoxy-ß-D-glucopyranosyl-(CH2)8COOCH3; HEPES, N-[2-hydroxyethyl]piperazine-N'-2-ethanesulfonic acid; PAPS, 3'-phosphoadenosine-5'-phosphosulfonate; PCR, polymerase chain reaction; POMC, proopiomelanocortin; RT–PCR, reverse transcriptase polymerase chain reaction
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