1 Institute for Molecular Science of Medicine, Aichi Medical University, 21 Yazako, Nagakute, Aichi 480-1195, Japan
2 Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA
* Author for correspondence (e-mail: kimata{at}amugw.aichi-med-u.ac.jp)
Accepted 26 February 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Heparan sulfate, Sulfotransferase, Golgi, Enzyme complex, Stem region
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 TransFASTTM 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.
Cell staining
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.
Inhibitor treatment
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 -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
).
Gel filtration
m6STsYQY-EGFP- or m6STsC2-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 SuperoseTM6 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 m6ST1C2-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 m6ST2
C2-EGFP and m6ST3
C2-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 (m6ST1
stem20-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
stem55 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
stem 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-m6ST1
CHO 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 1, 3-galactosyltransferase and
1, 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 ß1, 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 proTNF and m6ST1-EGFP in which the flanking region between the transmembrane domain and mature TNF
was replaced by the stem domain of HS6ST1 [proTNF
(m6ST1stem)-EGFP]. ProTNF
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 m6STstem20-EGFP and m6ST
stem55-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 pm6ST1
C2-EGFP or pm6ST1YQY-EGFP, which differed only in the presence or absence of the stem region. When the cell lysates of m6ST1
C2-EGFP-transfected COS7 cells were applied onto SuperoseTM6, 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.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 m6STsC2-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 (m6ST1
stem20-EGFP and m6ST1
stem55-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 proTNF
, a type II transmembrane protein that localizes primarily to the plasma membrane, the resultant chimeric protein [proTNF
(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 m6ST1stem20-EGFP and m6ST1
stem55-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
2,6-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 m6ST1stem20-EGFP and m6ST1
stem55-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.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashikari-Hada, S., Habuchi, H., Itoh, N., Reddi, A. H. and Kimata, K. (2004). Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library. J. Biol. Chem. 279, 12346-12354.
Bai, X. and Esko, J. D. (1996). An animal cell mutant defective in heparan sulfate hexuronic acid 2-O-sulfation. J. Biol. Chem. 271, 17711-17717.
Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L. and Lose, E. J. (1992). Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu. Rev. Cell Biol. 8, 365-393.[CrossRef][Medline]
Blackhall, F. H., Merry, C. L., Lyon, M., Jayson, G. C., Folkman, J., Javaherian, K. and Gallagher, J. T. (2003). Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates. Biochem. J. 375, 131-139.[CrossRef][Medline]
Brickman, Y. G., Ford, M. D., Gallagher, J. T., Nurcombe, V., Bartlett, P. F. and Turnbull, J. E. (1998). Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development. J. Biol. Chem. 273, 4350-4359.
Bullock, S. L., Fletcher, J. M., Beddington, R. S. and Wilson, V. A. (1998). Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase. Genes Dev. 12, 1894-1906.
Chen, C., Ma, J., Lazic, A., Backovic, M. and Colley, K. J. (2000). Formation of insoluble oligomers correlates with ST6Gal I stable localization in the golgi. J. Biol. Chem. 275, 13819-13826.
Colley, K. J. (1997). Golgi localization of glycosyltransferases: more questions than answers. Glycobiology 7, 1-13.[Abstract]
Crawford, B. E., Olson, S. K., Esko, J. D. and Pinhal, M. A. (2001). Cloning, golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid c5-epimerase. J. Biol. Chem. 276, 21538-21543.
Delehedde, M., Seve, M., Sergeant, N., Wartelle, I., Lyon, M., Rudland, P. S. and Fernig, D. G. (2000). Fibroblast growth factor-2 stimulation of p42/44MAPK phosphorylation and IkappaB degradation is regulated by heparan sulfate/heparin in rat mammary fibroblasts. J. Biol. Chem. 275, 33905-33910.
Ellgaard, L. and Helenius, A. (2003). Quality control in the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 4, 181-191.[CrossRef][Medline]
Esko, J. D. and Lindahl, U. (2001). Molecular diversity of heparan sulfate. J. Clin. Invest. 108, 169-173.
Fan, G., Xiao, L., Cheng, L., Wang, X., Sun, B. and Hu, G. (2000). Targeted disruption of NDST-1 gene leads to pulmonary hypoplasia and neonatal respiratory distress in mice. FEBS Lett. 467, 7-11.[CrossRef][Medline]
Ford-Perriss, M., Guimond, S. E., Greferath, U., Kita, M., Grobe, K., Habuchi, H., Kimata, K., Esko, J. D., Murphy, M. and Turnbull, J. E. (2002). Variant heparan sulfates synthesized in developing mouse brain differentially regulate FGF signaling. Glycobiology 12, 721-727.
Forsberg, E. and Kjellen, L. (2001). Heparan sulfate: lessons from knockout mice. J. Clin. Invest. 108, 175-180.
Giraldez, A. J., Copley, R. R. and Cohen, S. M. (2002). HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient. Dev. Cell 2, 667-676.[Medline]
Habuchi, H., Suzuki, S., Saito, T., Tamura, T., Harada, T., Yoshida, K. and Kimata, K. (1992). Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor. Biochem. J. 285, 805-813.[Medline]
Habuchi, H., Habuchi, O. and Kimata, K. (1995). Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells. J. Biol. Chem. 270, 4172-4179.
Habuchi, H., Tanaka, M., Habuchi, O., Yoshida, K., Suzuki, H., Ban, K. and Kimata, K. (2000). The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine. J. Biol. Chem. 275, 2859-2868.
Habuchi, H., Miyake, G., Nogami, K., Kuroiwa, A., Matsuda, Y., Kusche-Gullberg, M., Habuchi, O., Tanaka, M. and Kimata, K. (2003). Biosynthesis of heparan sulphate with diverse structures and functions: two alternatively spliced forms of human heparan sulphate 6-O-sulphotransferase-2 having different expression patterns and properties. Biochem. J. 371, 131-142.[CrossRef][Medline]
Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y. and Noda, M. (1993). Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes. J. Biol. Chem. 268, 21968-21974.
Hampton, R. Y. (2002). ER-associated degradation in protein quality control and cellular regulation. Curr. Opin. Cell Biol. 14, 476-482.[CrossRef][Medline]
Humphries, D. E., Sullivan, B. M., Aleixo, M. D. and Stow, J. L. (1997). Localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase to the trans-Golgi network. Biochem. J. 325, 351-357.[Medline]
Kamimura, K., Fujise, M., Villa, F., Izumi, S., Habuchi, H., Kimata, K. and Nakato, H. (2001). Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST) gene. Structure, expression, and function in the formation of the tracheal system. J. Biol. Chem. 276, 17014-17021.
Kan, M., Wang, F., Xu, J., Crabb, J. W., Hou, J. and McKeehan, W. L. (1993). An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science 259, 1918-1921.[Medline]
Kjellen, L. and Lindahl, U. (1991). Proteoglycans: structures and interactions. Annu. Rev. Biochem. 60, 443-475.[CrossRef][Medline]
Kobayashi, S., Morimoto, K., Shimizu, T., Takahashi, M., Kurosawa, H. and Shirasawa, T. (2000). Association of EXT1 and EXT2, hereditary multiple exostoses gene products, in Golgi apparatus. Biochem. Biophys. Res. Commun. 268, 860-867.[CrossRef][Medline]
Lin, X., Wei, G., Shi, Z., Dryer, L., Esko, J. D., Wells, D. E. and Matzuk, M. M. (2000). Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice. Dev. Biol. 224, 299-311.[CrossRef][Medline]
Lippincott-Schwartz, J., Yuan, L. C., Bonafacino, J. S. and Klausner, R. D. (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801-813.[Medline]
Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C. and Klausner, R. D. (1990). Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60, 821-836.[Medline]
Liu, J., Shworak, N. W., Fritze, L. M., Edelberg, J. M. and Rosenberg, R. D. (1996). Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 271, 27072-27082.
Masibay, A. S., Balaji, P. V., Boeggeman, E. E. and Qasba, P. K. (1993). Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase. J. Biol. Chem. 268, 9908-9916.
McCormick, C., Duncan, G., Goutsos, K. T. and Tufaro, F. (2000). The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate. Proc. Natl. Acad. Sci. USA 97, 668-673.
Merry, C. L., Bullock, S. L., Swan, D. C., Backen, A. C., Lyon, M., Beddington, R. S., Wilson, V. A. and Gallagher, J. T. (2001). The molecular phenotype of heparan sulfate in the Hs2st/ mutant mouse. J. Biol. Chem. 276, 35429-35434.
Milland, J., Russell, S. M., Dodson, H. C., McKenzie, I. F. and Sandrin, M. S. (2002). The cytoplasmic tail of alpha 1,3-galactosyltransferase inhibits Golgi localization of the full-length enzyme. J. Biol. Chem. 277, 10374-10378.
Nakamura, N., Rabouille, C., Watson, R., Nilsson, T., Hui, N., Slusarewicz, P., Kreis, T. E. and Warren, G. (1995). Characterization of a cis-Golgi matrix protein, GM130. J. Cell Biol. 6, 1715-1726.[CrossRef]
Nilsson, T., Hoe, M. H., Slusarewicz, P., Rabouille, C., Watson, R., Hunte, F., Watzele, G., Berger, E. G. and Warren, G. (1994). Kin recognition between medial Golgi enzymes in HeLa cells. EMBO J. 13, 562-574.[Abstract]
Nilsson, T., Rabouille, C., Hui, N., Watson, R. and Warren, G. (1996). The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. J. Cell Sci. 109, 1975-1989.
Opat, A. S., Houghton, F. and Gleeson, P. A. (2000). Medial Golgi but not late Golgi glycosyltransferases exist as high molecular weight complexes. Role of luminal domain in complex formation and localization. J. Biol. Chem. 275, 11836-11845.
Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derymck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. and Goeddel, D. V. (1984). Human tumor necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 312, 724-729.[Medline]
Pinhal, M. A., Smith, B., Olson, S., Aikawa, J., Kimata, K. and Esko, J. D. (2001). Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo. Proc. Natl. Acad. Sci. USA 98, 12984-12989.
Ringvall, M., Ledin, J., Holmborn, K., van Kuppevelt, T., Ellin, F., Eriksson, I., Olofsson, A. M., Kjellen, L. and Forsberg, E. (2000). Defective heparan sulfate biosynthesis and neonatal lethality in mice lacking N-Deacetylase/N-sulfotransferase-1. J. Biol. Chem. 275, 25926-25930.
Saraste, J. and Svensson, K. (1991). Distribution of the intermediate elements operating in ER to Golgi transport. J. Cell Sci. 100, 415-430.[Abstract]
Sasai, K., Ikeda, Y., Tsuda, T., Ihara, H., Korekane, H., Shiota, K. and Taniguchi, N. (2001). The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction. J. Biol. Chem. 276, 759-765.
Smeds, E., Habuchi, H., Do, A. T., Hjertson, E., Grundberg, H., Kimata, K., Lindahl, U. and Kusche-Gullberg, M. (2003). Substrate specificities of mouse heparan sulphate glucosaminyl 6-O-sulphotransferases. Biochem. J. 372, 371-380.[CrossRef][Medline]
Teasdale, R. D., D'Agostaro, G. and Gleeson, P. A. (1992). The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain. J. Biol. Chem. 267, 4084-4096.
Teasdale, R. D., Matheson, F. and Gleeson, P. A. (1994). Post-translational modifications distinguish cell surface from Golgi-retained beta 1,4 galactosyltransferase molecules. Golgi localization involves active retention. Glycobiology 4, 917-928.[Abstract]
Turnbull, J., Powell, A. and Guimond, S. (2001). Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol. 11, 75-82.[CrossRef][Medline]
Utsumi, T., Akimaru, K., Kawabata, Z., Levitan, A., Tokunaga, T., Tang, P., Ide, A., Hung, M. C. and Klostergaard, J. (1995). Human pro-tumor necrosis factor: molecular determinants of membrane translocation, sorting, and maturation. Mol. Cell. Biol. 15, 6398-6405.[Abstract]
Yang, W. and Storrie, B. (1998). Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins. Mol. Biol. Cell 9, 191-207