Heparan sulfate O-sulfotransferases catalyze the O-sulfation of the glucosamine and uronic acid residues of heparan sulfate, thereby determining the binding sites for ligands necessary for important biological functions such as the formation of morphogen gradients and growth factor signaling. Here we investigated the localization of the three heparan sulfate 6-O-sulfotransferase (HS6ST) isoforms and the mechanism of their localization. All three GFP-tagged HS6STs localized in the Golgi apparatus. C-5 epimerase and HS2ST have been shown to form complexes that facilitate their localization in the Golgi but we found that the absence of HS2ST did not alter the localization of any of the HS6STs. Neither the forced expression of HS2ST in the rough endoplasmic reticulum (ER), the deletion of most of the lumenal domain nor increasing the length of the transmembrane domain had any effect on the localization of HS6STs. However, deletions in the stem region did affect the Golgi localization of the HS6STs and also reduced their sulfotransferase activity and oligomer formation. These findings suggest that the stem region of HS6ST plays an important role in normal functioning, including the transit of HS6ST to the Golgi apparatus and maintaining the active conformation essential for enzyme activity.
Heparin and heparan sulfate (HS) proteoglycans play important roles in many biological processes, including the regulation of blood coagulation, cell recognition, adhesion and differentiation, and proliferation mediated by a variety of growth factors and morphogens (Bernfield et al., 1992; Delehedde et al., 2000; Giraldez et al., 2002; Kjellen and Lindahl, 1991; Turnbull et al., 2001). The functions of heparin and HS proteoglycans in these various biological processes are dependent upon the specific arrangements of their sulfated sugars and uronic acids that make up the binding sites for particular molecules (Blackhall et al., 2003; Esko and Lindahl, 2001; Ford-Perriss et al., 2002; Ashikari-Hada et al., 2004). For example, N-deacetylation and N-sulfation of N-acetylglucosamine (GlcNAc) residues and 2-O-sulfation of adjacent iduronic acid (IdoA) residues are important for the binding of basic fibroblast growth factor (Habuchi et al., 1992; Brickman et al., 1998). Further 6-O-sulfation of the N-sulfated glucosamine residues appears to be essential for the subsequent signaling (Kan et al., 1993). Abnormalities found in animals that lack the enzymes catalyzing these modifications have reinforced the idea that heparin/HS modification plays a pivotal role (for a review, see Forsberg and Kjellen, 2001). Mice lacking N-deacetylase/N-sulfotransferase (NDST-1) have forebrain defects and develop respiratory distress and atelectasis that lead to neonatal death (Fan et al., 2000). Mice defective in heparan sulfate 2-sulfotransferase (HS2ST) also die perinatally, and they exhibit bilateral renal agenesis and defects of the eye and skeleton, possibly because of abnormal fibroblast growth factor (FGF) and Wnt signaling (Bullock et al., 1998; Merry et al., 2001). RNA interference experiments in Drosophila have also demonstrated that reduction of heparan sulfate 6-sulfotransferase (dHS6ST) activity leads to a lethal phenotype due to the inhibition of the FGF signaling pathway (Kamimura et al., 2001). Thus, the fine structures of HS created by specific O-sulfation reactions are essential for normal development and morphogenesis (Lin et al., 2000; Ringvall et al., 2000).
Heparin and HS are synthesized by polymerizing alternating glucuronic acid (GlcA) and GlcNAc residues to the linkage tetrasaccharide GlcA-galactose(Gal)-Gal-xylose, which is attached to Ser residues of proteoglycan core proteins (Esko and Lindahl, 2001). Simultaneously, a series of modification reactions takes place. These include N-deacetylation and N-sulfation of GlcNAc residues, C-5 epimerization of GlcA to IdoA residues, 2-O-sulfation of IdoA residues (and occasionally GlcA residues), and 6-O- and 3-O-sulfation of glucosamine residues. All the enzymes involved in the biosynthesis of heparin and HS chains that have been reported to date appear to reside in the Golgi complex (Crawford et al., 2001; Humphries et al., 1997). EXT1 and EXT2, the copolymerase complex, reside in the Golgi apparatus (Kobayashi et al., 2000; McCormick et al., 2000), as HS2ST and C-5 epimerase (Pinhal et al., 2001). Presumably, the precise localization of these enzymes in the membranes of the Golgi subcompartments plays a role in the organized synthesis of heparin/HS chains. The reported observations also suggest that some of the biosynthetic enzymes assemble into macromolecular complexes that facilitate interactions with substrates and control their enzymatic activity.
We have recently shown that the HS6STs belong to a gene family composed of three isoforms (HS6ST-1, -2, and -3) and an alternatively spliced form of HS6ST-2 (HS6ST-2S) (Habuchi et al., 2003; Smeds et al., 2003). These are type II transmembrane proteins that have subtle but significant differences in substrate specificity. Although biochemical studies have suggested that 6-O-sulfation occurs as a rather late biosynthetic event (Habuchi et al., 2000), the localization of the HS6ST isoforms in the Golgi apparatus and their interactions with other enzymes have not been studied. Here, we examine the distribution and activities of green fluorescent protein (GFP)-tagged forms of these enzymes and their deletion or insertion mutants. We show that the stem regions of the HS6STs play an important role in their normal functioning as these regions promote the localization to the Golgi apparatus. The activity of the stem region is supported by the cytoplasmic and/or transmembrane domains but not by the lumenal domains. In addition, we show that deletions in the stem region not only affect the localization of the HS6ST isoforms, but also reduce the sulfotransferase activity of the enzymes and their oligomerization. This suggests that the stem region maintains an active conformation essential for enzyme activity.
Materials and Methods
The GFP expression vector pEGFP-N1 was purchased from Clontech. The plasmids used in this study were constructed by polymerase chain reaction (PCR)-based mutagenesis except for pm2ST-EGFP, pm6ST1-EGFP, pm6ST2-EGFP and pm6ST3-EGFP. The coding regions of the mouse HS2ST and HS6ST genes were excised from FLAG-tagged expression vectors (Habuchi et al., 2000) and subcloned as depicted in Table 1. The primers and plasmids used in the PCR reactions are listed in Table 1. Briefly, PCR was performed using forward and reverse primer sets and the Expand™ Long Template PCR system (Roche) as described in the manufacturer's protocol. Blunt ends were created on the PCR products with T4-polymerase (New England Biolabs) and the products were treated with T4 polynucleotide kinase (New England Biolabs), ligated, and transformed into competent Escherichia coli cells. Drug-resistant colonies were selected for DNA isolation and the inserts of their plasmids were confirmed by sequencing. If necessary, the plasmids were further subcloned using appropriate plasmids and restriction enzyme sites. The plasmid encoding human pro-tumor necrosis factor α (TNFα) cDNA (pUTNF2) (Pennica et al., 1984) was provided by Dr N. Watanabe (Toho University). The coding region was excised with EcoRI and subcloned into the EcoRI site of pEGFP-N1 (pproTNFαstop-EGFP) and then used to construct pproTNFα(m6ST1stem)-EGFP. The predicted protein structures encoded by the plasmids are shown schematically in the figures.
Cell culture and transfection
DNA was purified using the QIAGEN midi or maxi kit and transfected into CHO-K1 or CHO-F17 cells using the TransFAST™ transfection reagent (Promega) as recommended by the manufacturer. The cells were grown in Dulbecco's modified Eagle medium/F-12 medium (Sigma) supplemented with 10% FCS.
Alexa Fluor secondary antibodies were purchased from Molecular Probes. Rabbit polyclonal anti-GM130 antiserum was provided by Dr N. Nakamura (Kanazawa University). Cells on cover slips were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 15 minutes. After washing with PBS several times, the cells were permeabilized with 0.2% Triton X-100 in PBS at room temperature for 1 minute. The cells were sequentially incubated with blocking solution (0.2% gelatin in PBS), anti-GM130 antiserum (1:200 dilution in 0.2% gelatin-PBS) or mouse anti-FLAG monoclonal antibody (M2, Sigma) (1:10,000 dilution in 0.2% gelatin-PBS) for 1 hour. They were then washed and further incubated for 1 hour with a ∼1:1000-2000 dilution of Alexa Fluor secondary antibody. Immunofluorescence was detected with an Olympus fluorescence microscope. In some cases, cells were fixed with 80% ethanol for 1 hour at 4°C to remove the cytosolic proteins. In the case of m6ST1TMmut-EGFP, the cells were incubated with 100 μM cycloheximide for 30, 90, or 180 minutes before fixation to chase out the newly synthesized protein. The cells were then fixed with paraformaldehyde or 80% ethanol as described above.
Brefeldin A (BFA) and nocodazole were purchased from Sigma. Cycloheximide was from Wako Chemicals. Cells cultured on coverslips were incubated with 100 μM cycloheximide for 30 minutes in the culture medium, then treated with 10 μg/ml BFA for 1 hour or 20 μM nocodazole for 1 or 2 hours in the presence of cycloheximide in the culture medium. Fixation and staining were performed as described above.
Sulfotransferase activity assay
Cells cultured in 60-mm plastic tissue culture dishes were scraped into 500 μl of ice-cold buffer A [10 mM Tris (pH 7.2), 2 mM CaCl2, 10 mM MgCl2, 150 mM NaCl, 0.5% Triton X-100, 20% glycerol, and a cocktail of protease inhibitors (5 μM N α-tosyl-L-lysine chloromethyl ketone, 3 μM L-1-p-tosylamino-2-phenylethyl chloromethyl ketone, 30 μM phenylmethyl-sulfonyl fluoride, 3 μM pepstatin)]. After rotating for 1 hour at 4°C, the crude cell lysate was clarified by centrifugation at 12,000 g for 20 minutes. To assay the FLAG-tagged protein, CHO-K1 cells were transfected with expression vectors and 24 hours later, cell lysates were prepared as described above. The cell lysates were incubated with a 15-μl bed volume of anti-FLAG M2 affinity gel (Sigma) overnight at 4°C. The gel was then washed several times with buffer A after which the bound protein was extracted with buffer A containing 125 μg/ml FLAG-peptide. Half of the samples were subjected to sodium lauryl sulfate-PAGE. Western blotting was performed using the anti-FLAG monoclonal antibody M5 (Sigma), and the band intensity was quantified with NIH Image software. The other half of the FLAG-tagged proteins were normalized as above and used for sulfotransferase assays. Sulfotransferase activity was measured as previously described (Habuchi et al., 1993). The standard reaction mixture contained 2.5 μmol imidazole HCl, pH 6.8; 3.75 μg protamine chloride; 25 nmol complete desulfated and N-resulfated-heparin; 50 pmol [35S] 3′-phosphoadenosine 5′-phosphosulfate (PAPS); and the protein or cell lysate obtained as described above in a final volume of 50 μl. The reaction mixtures were incubated at 37°C for 20 minutes and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 minute. Chondroitin sulfate A (0.1 μmol as glucuronic acid) was added to the reaction mixture as a carrier. 35S-labeled polysaccharides were precipitated with 3 volumes of cold ethanol containing 1.3% potassium acetate and separated completely from [35S]PAPS and its degradation products by gel chromatography using Fast Desalting columns as described previously (Habuchi et al., 1993).
m6STsYQY-EGFP- or m6STsΔC2-EGFP-transfected COS7 cells cultured in 60 mm plastic tissue culture dishes were scraped into 500 ml of ice-cold buffer A. After rotating for 1 hour at 4°C, the crude cell lysate was clarified by centrifugation at 12,000 g for 20 minutes. The extracts were fractionated immediately by HR10/30 Superose™6 fast protein liquid chromatography (FPLC)(Amersham Pharmacia Biotech). Loading and elution of the FPLC system was carried out with buffer A (without glycerol) at 0.5 ml/minute. After waiting for 12 minutes, 0.5-ml fractions were collected and the GFP fluorescence of each fraction was measured using fluorescent spectrophotometer (HITACHI, F-3010) at the excitation wavelength 488 nm and the emission wavelength 507 nm.
Golgi localization of the HS6ST proteins
We first investigated the subcellular localization of GFP-tagged mouse HS6ST-1, -2, and -3 (m6STs) in transfected CHO-K1 and COS7 cells (Fig. 1A). All proteins exhibited sulfotransferase activity at levels similar to that of their FLAG-tagged proteins (data not shown), as reported previously (Habuchi et al., 2000), which suggests that these recombinant proteins were in their proper conformation. When expressed in CHO-K1 cells, all the proteins co-localized with the Golgi marker GM130 (Fig. 1B) but not with the ER marker protein disulfide isomerase (data not shown). Similar results were obtained with COS7 cells (data not shown).
BFA and nocodazole are known to disrupt the structure of the Golgi apparatus by different mechanisms. BFA prevents ER-to-Golgi trafficking and causes the Golgi apparatus to tubulate. Most of the Golgi proteins are redistributed into the ER but the marker proteins in the cis-Golgi network are not affected (Lippincott-Schwartz et al., 1989; Lippincott-Schwartz et al., 1990; Saraste and Svensson, 1991). Nocodazole treatment, in contrast, causes the Golgi apparatus to fragment into large vesicles at the cell periphery by disrupting the microtubule network. The scattering of the cis-side Golgi proteins lags behind the scattering of the trans-side proteins (Yang and Storrie, 1998). In BFA-treated cells, the cis-Golgi protein GM130 was found in dispersed and punctate structures (Fig. 1C) as has been shown previously (Nakamura et al., 1995). In contrast, the m6ST1-EGFP protein was redistributed to the ER in characteristic fine tubular-like structures. This is consistent with its localization in the medial- or trans-Golgi. As shown in Fig. 1C, the scattering of the m6ST1-EGFP protein upon nocodazole treatment preceded than that of the cis-Golgi marker GM130, which suggests that m6ST1-EGFP is localized more at the trans side than GM130 (compare the vesicular structures of m6ST1-EGFP and GM130 at 1 and 2 hours). The m6ST2-EGFP and m6ST3-EGFP proteins showed similar results (data not shown). Therefore, all three HS6STs reside in the trans-side of the Golgi stack.
Localization of the HS6ST proteins in the absence of endogenous HS2ST protein
The formation of a complex between EXT1 and EXT2 and the physical association of C-5 epimerase and HS2ST are important for their respective activities and facilitates their localization in the Golgi apparatus (Kobayashi et al., 2000; McCormick et al., 2000; Pinhal et al., 2001). Therefore, we examined whether the HS6STs interact with other enzymes in the pathway. First, we expressed the GFP-tagged HS6ST proteins in the HS2ST-deficient CHO-K1-derived cell line CHO-F17 (Bai and Esko, 1996) and examined their localization. All GFP-tagged HS6STs (m6STs-EGFP) localized normally to the Golgi in the CHO-F17 cells (Fig. 2A). Furthermore, expression of a recombinant form of HS2ST that is forced to localize to the ER (FLAG/p33/m2ST) (Fig. 2B, upper panel) did not disturb the Golgi localization of the GFP-tagged HS6ST1 protein (Fig. 2C). In the latter experiment, we used a retargeting method in which an ER retention peptide from the human invariant chain (hIip33) is grafted onto the N terminus of transferases (Nilsson et al., 1994). The GFP-tagged HS6ST2 and HS6ST3 proteins yielded the same results (data not shown). Similarly, expression of the FLAG-tagged ER-localized HS6ST1 protein (FLAG/p33/m6ST1) (Fig. 2B, upper panel) did not affect the Golgi localization of HS2ST-GFP (Fig. 2C). FLAG/p33-tagged HS6ST2 and HS6ST3 also gave the same results (data not shown). All the ER-localized HS2ST (FLAG/p33/m2ST) and HS6ST (FLAG/p33/m6STs) proteins showed sulfotransferase activities comparable to those of the Golgi-localized enzymes (FLAG-m2ST and the FLAG-m6STs) (Fig. 2B, right panel). This argues against the possibility that the FLAG/p33/m2ST and the FLAG/p33/m6STs were in abnormal conformation blocking protein interactions.
Roles of the stem domains in Golgi localization of HS6STs
We next investigated which domains of the HS6ST proteins are responsible for their Golgi localization. First, we considered whether the Golgi localization signal resides in the lumenal domain. Mutant HS6ST1 proteins bearing various C-terminal deletions were introduced into CHO-K1 (Fig. 3A) and analyzed by fluorescence microscopy. As shown with m6ST1ΔC2-EGFP in Fig. 3A, a mutant HS6ST1 protein that consists of the N-terminal cytoplasmic tail, the transmembrane domain, and the 55-amino-acid stem region still localized in the Golgi. The m6ST2ΔC2-EGFP and m6ST3ΔC2-EGFP proteins, whose putative stem domains are 58 and 123 amino acids long, respectively, gave similar results (data not shown). However, deleting the first 20 amino acids of the membrane-proximal portion of the lumenal domain (m6ST1Δstem20-EGFP) inhibited Golgi localization (Fig. 3A), and this inhibition was complete when the entire 55 amino acids of the stem region were deleted (see the Δstem55 mutant in Fig. 3A). Similar results were obtained with the other m6ST isozymes (data not shown). In addition, a mutant EGFP-tagged HS6ST1 protein consisting of the N-terminal cytoplasmic tail, the transmembrane domain, and three amino acids of the lumenal region (m6ST1YQY-EGFP) localized in the cytoplasm (Fig. 3A). These results clearly demonstrate that the stem region is important for the Golgi targeting of the HS6ST isozymes. However, truncating the stem region to the first 20 membrane-proximal amino acids did not compromise the Golgi localization (see m6ST1stem20-EGFP in Fig. 3B for a representative example).
It is well known that misfolded proteins are sequestered in the ER for degradation by the quality control mechanism (Ellgaard and Helenius, 2003; Hampton, 2002). Therefore, the mislocalization of the Δstem mutants shown above could be due to their aberrant conformation. However, an enzymatically inactive and perhaps misfolded form of a HS6ST-1 mutant in which two Asn residues in the N-glycosylation site had been mutated to Gln (the FLAG-m6ST1ΔCHO mutant) co-localized with the Golgi marker GM130 (data not shown).
Although the amino acid sequences of the membrane-proximal portion of the stem regions of the three HS6ST proteins are divergent, the Gln24, Tyr25, and Pro28 residues are well conserved. We focused on the former two (Gln24 and Tyr25) as all HS6STs are proteolytically processed between Gln24 and Tyr25 so that the lumenal domains can be secreted into the medium (Habuchi et al., 1995). Mutation of both these residues to Ala [m6ST1stem20(AA)-EGFP] did not affect the Golgi localization profile (Fig. 3B).
It has been shown that the cytoplasmic tails of the Golgi-resident enzymes α1, 3-galactosyltransferase and α1, 2-fucosyltransferase are sufficient to confer their specific Golgi localization (Milland et al., 2002). Of the seven amino acids in the cytoplasmic tails of mouse HS6ST-1, -2, and -3, three residues (Asp3, Arg4 or Lys4, and Lys7) are conserved. Mutating each or all (MVAAASA) of these residues to alanine did not change the localization profile (Fig. 3B and data not shown).
The length of the transmembrane domain is an important parameter for the Golgi retention of β1, 4-galactosyltransferase (Teasdale et al., 1992), which is consistent with a model in which the bilayer thickness plays a role (Teasdale et al., 1994; Utsumi et al., 1995). We changed the length of the transmembrane domain in m6ST1stem20-EGFP by replacing it with that of the plasma membrane protein pro-tumor necrosis factor (pro-TNF) (m6ST1TMmutstem20-EGFP) (Nilsson et al., 1996). The recombinant protein m6ST1TMmutstem20-EGFP has a transmembrane domain that is 11 amino acids longer than that of m6ST1stem20-EGFP. When we used this construct, GFP fluorescence in paraformaldehyde-fixed cells was detected all over the cells, with punctate staining around the nucleus (Fig. 3B). When cells fixed with ethanol were used, the cytosolic staining disappeared but the perinuclear punctate staining was still observed (Fig. 3B). This indicates that the GFP protein had been located in the Golgi apparatus. Therefore, the increased length of the hydrophobic region did not totally compromise the localization of HS6ST1.
We finally examined whether the presence of the stem region is sufficient for Golgi localization. To do this, we generated a chimeric protein of proTNFα and m6ST1-EGFP in which the flanking region between the transmembrane domain and mature TNFα was replaced by the stem domain of HS6ST1 [proTNFα(m6ST1stem)-EGFP]. ProTNFα was chosen because it also has type-II transmembrane topology like HS6ST but localizes mainly at the cell surface. The fluorescence of this chimeric protein was also observed at the perinucleus. However, this molecule did not co-localize with the Golgi marker GM130. This result strongly indicates that domains other than the stem region support the localization of HS6STs to the Golgi apparatus.
Stem region is also important for enzyme activity and complex formation
To examine whether the stem region is involved in the enzyme activity of the HS6ST proteins, we assayed the sulfotransferase activity of the m6STΔstem20-EGFP and m6STΔstem55-EGFP mutants. The larger deletion in m6ST1 resulted in a greater reduction of sulfotransferase activity, which demonstrates its role in conferring enzyme activity (Fig. 4A). Similar results were obtained for the other two isoforms (data not shown).
It has been shown that the stem region of N-acetylglucosaminyltransferase V (GnT-V) is responsible not only for its intracellular localization but also for its oligomerization (Sasai et al., 2001). We tested the possibility of HS6ST complex formation by gel filtration using COS7 cells transfected with pm6ST1ΔC2-EGFP or pm6ST1YQY-EGFP, which differed only in the presence or absence of the stem region. When the cell lysates of m6ST1ΔC2-EGFP-transfected COS7 cells were applied onto Superose™6, GFP-fluorescent peaks corresponding to the monomer and oligomer appeared (Fig. 4B). As the oligomer peak is rather small, one could propose that the Triton X-100 treatment to obtain the cell lysate is harsh and disrupts some oligomers. In contrast, the elution profile of m6ST1YQY-EGFP showed only one peak corresponding to the monomer (Fig. 4B). Similar results were obtained with the corresponding mutants of the HS6ST-2 and -3 isoforms. This indicates that the stem domains of all three HS6ST isoforms promote the formation of oligomers. Together, these results suggest that the membrane-proximal stem domain not only plays an essential role in the Golgi localization of the HS6STs, but that it also ensures their enzyme activity and oligomerization.
Golgi localization of the HS6ST proteins
In this paper, we demonstrated that the HS6STs are Golgi-resident enzymes because when introduced into CHO-K1 cells, all GFP-tagged isoforms of the HS6STs co-localized with the Golgi marker GM130 (Fig. 1B). Two lines of evidence obtained in the present study demonstrate that the three isoforms of HS6ST do not require HS2ST for their Golgi localization. First, in the CHO-F17 cell line that lacks endogenous HS2ST expression, the three isoforms of HS6ST still localized to the Golgi apparatus (Fig. 2A). Second, expression in CHO-K1 cells of HS2ST that is forced to localize to the ER (FLAG/p33/m2ST) together with the GFP-tagged HS6ST proteins (m6STs-EGFP) did not alter the Golgi-staining pattern of GFP, and vice versa (Fig. 2C). This contrasts with the observations that EXT1 and EXT2, and HS2ST and C-5 epimerase are partly dependent on each other for localization to the Golgi apparatus (Pinhal et al., 2001).
Importance of the stem domain of the HS6ST proteins
Mutant HS6ST proteins that consist only of the N-terminal cytoplasmic tail, the transmembrane domain, and the stem region showed efficient Golgi localization (see m6STsΔC2-EGFP in Fig. 3A). This indicates that these three domains are sufficient for the Golgi localization of the HS6ST proteins. Truncating the stem region to the membrane-proximal 20 amino acids also did not compromise the Golgi localization (see m6ST1stem20-EGFP in Fig. 3B). We mutated several residues in the stem region or the cytoplasmic tail showing conservation between the three HS6STs, but none of these mutations affected the efficient Golgi retention of the proteins (Fig. 3B). However, complete deletion of the stem region impaired the localization of the HS6STs (m6ST1Δstem20-EGFP and m6ST1Δstem55-EGFP in Fig. 3A), which shows that it plays a significant role in Golgi localization. Similar results have been reported for GnT-V, a Golgi-resident protein whose stem region is not required for its enzymatic activity but is responsible for its intracellular localization (Sasai et al., 2001). However, when we transferred the HS6ST1 stem region to proTNFα, a type II transmembrane protein that localizes primarily to the plasma membrane, the resultant chimeric protein [proTNFα(m6ST1stem)-EGFP] did not localize to the Golgi (Fig. 3B). These results suggest that the stem regions of the HS6STs are not the sole determinants for their Golgi localization. It is likely that the cytoplasmic and/or transmembrane regions act in combination with the stem region to guide the HS6STs to the Golgi. Thus, the membrane-proximal portion of the stem region in all three HS6STs plays a central role in their Golgi localization that is supported by the cytoplasmic and/or transmembrane regions.
When we increased the length of the HS6ST1 transmembrane region from 15 amino acids to 26 amino acids, the tagged protein was detected in both the cytosol and the Golgi (Fig. 3B). The cytosolic location of some of the m6ST1TMmut-EGFP may be due to inefficient translocation of the fusion protein across the ER membrane. However, the fact that some of the mutant protein still localized in the Golgi apparatus emphasizes the importance of the stem region rather than the transmembrane domain in targeting these molecules to the Golgi.
Deleting the membrane-proximal portion of the stem region may affect the conformation of the lumenal domain as the sulfotransferase activities of m6ST1Δstem20-EGFP and m6ST1Δstem55-EGFP were reduced (Fig. 4A). As misfolded proteins are sequestered in the ER by the quality control mechanism (Ellgaard and Helenius, 2003; Hampton, 2002), the mislocalization of the mutants lacking the stem region could be due to an aberrant conformation. However, we found that an HS6ST-1 mutant lacking N-glycosylation sites that is enzymatically inactive and perhaps also misfolded, still nevertheless co-localized with the Golgi marker GM130 (data not shown). This suggests that the stem region may participate in the Golgi localization of HS6ST proteins in ways other than simply maintaining the conformation in an active state.
Interactions with other Golgi-resident enzymes is one possible mechanism by which the stem region promotes HS6ST localization in the Golgi apparatus (Masibay et al., 1993) supported by the gel filtration studies showing that only the HS6ST protein bearing the stem region can oligomerize (Fig. 4B). Such enzyme complex formation may be responsible for the Golgi localization of the HS6STs, as has been found to be the case for N-acetylglucosaminyltransferase I, N-acetylglucosaminyltransferase II, and α2,6-sialyltransferase (Chen et al., 2000; Colley, 1997; Opat et al., 2000). However, we could not determine whether the HS6STs reside in the Golgi apparatus by the kin recognition mechanism because HS6ST mutants that show varying oligomerizing potentials are not available. Nevertheless, our results suggest that the stem region is the principal domain that controls the Golgi localization of the HS6ST isoforms and affects their oligomer formation.
The stem region may also be important for efficient translocation. The mutants that lack the lumenal domain (m6STsYQY-EGFP), in which the cytoplasmic and transmembrane domains of the m6STs are connected directly to EGFP, did not translocate even to the ER and resulted in cytoplasmic fluorescence (Fig. 3A and data not shown). Given that m6ST1Δstem20-EGFP and m6ST1Δstem55-EGFP exhibited reduced enzymatic activity, the stem region may also act as a spacer that allows the catalytic domain to fold correctly. In this regard, a homologous sequence in the stem regions of the all HS6ST isoforms would not be necessary, which may explain why there is so little homology between their stem regions. We have also demonstrated in this paper that the stem domain promotes oligomer formation (Fig. 4B). We could not determine whether the stem domain participates in oligomer formation directly or indirectly by affecting the conformation of the transmembrane or cytoplasmic domains. It is also possible that these domains interact separately with other proteins. Another important role of the stem domain of HS6STs but not of HS2ST may relate to the shedding of the enzyme, as HS6ST activity but not HS2ST activity is detected in the culture medium (Habuchi et al., 1995). This difference was also observed between HS3ST (Liu et al., 1996) and the NDSTs, of which only the former activity is detected in the culture medium. Thus, the stem domains of the sulfotransferases that act on HS earlier (NDST and HS2ST) or later (HS6ST and HS3ST) may also regulate the shedding by distinct mechanisms.
In conclusion, we have shown that the HS6STs are Golgi-resident enzymes. HS2ST was not required for their localization in the Golgi. The stem regions were indispensable for their Golgi localization. We also demonstrated that the stem region is important for HS6ST enzyme activity and it also promotes oligomer formation. Therefore, modifying the stem region would give us a chance to control the activity of HS6STs, which would affect the levels of HS sulfation achieved by these enzymes.
We thank N. Nakamura for helpful suggestions. This work was supported by a Grant-in-aid for Scientific Research on Priority Areas 14082206, by a preparatory grant for research from the Division of Matrix Glycoconjugates, Research Center for Infectious Disease, Aichi Medical University; and by a special research fund from Seikagaku Corporation.
- Accepted February 26, 2004.
- © The Company of Biologists Limited 2004