2 Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
3 Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542, Japan
4 Meiji Dairies Corporation, 540 Naruda, Odawara 250-0862, Japan
Received on July 22, 2002; revised on August 16, 2002; accepted on August 18, 2002
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Abstract |
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Key words: chondroitin sulfate / heparan sulfate / sulfotransferase
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Introduction |
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Midkine (MK) is a heparin-binding growth factor structurally unrelated to FGFs (Iwasaki et al., 1997; Kadomatsu et al., 1988
; Muramatsu, 2002
; Muramatsu et al., 1993
; Owada et al., 1999
; Tomomura et al., 1990
) and together with heparin-binding growth-associated molecule (HB-GAM; also called pleiotrophin) (Li et al., 1990
; Merenmies and Rauvala, 1990
; Rauvala, 1989
) forms a new family of heparin-binding growth factors. MK promotes neurite outgrowth (Muramatsu et al., 1993
; Muramatsu and Muramatsu, 1991
), survival (Owada et al., 1999
), growth (Muramatsu and Muramatsu, 1991; Takei et al., 2001
), and migration (Horiba et al., 2000
; Maeda et al., 1999
; Qi et al., 2001
; Takada et al., 1997
) of various cells and is involved in tumor growth (Takei et al., 2001
), vascular diseases (Horiba et al., 2000
), and nephritis induced by ischemia (Sato et al., 2001
).
Interaction with the heparin-like domain in heparan sulfate proteoglycans is also important for the function of MK and HB-GAM. Thus, neurite outgrowth promotion by these factors is specifically inhibited by a low dose of heparin (Kaneda et al., 1996b; Kinnunen et al., 1996
). The C-terminal half of MK, which has a conformation-dependent heparin binding site, can promote neurite outgrowth (Muramatsu et al., 1994
). Furthermore, N-syndecan (syndecan-3) has been identified as the receptor or coreceptor of HB-GAM-induced neurite outgrowth of embryonic brain neurons (Raulo et al., 1994
). Recently, a chondroitin sulfate (CS) proteoglycan, receptor-type protein tyrosine phosphatase
, has been found as a receptor of MK (Maeda et al., 1999
; Qi et al., 2001
) and HB-GAM (Maeda and Noda, 1998
) in migration of embryonic neurons (Maeda et al., 1999
; Maeda and Noda, 1998
) and osteoblasts (Qi et al., 2001
). MK binds to the CS portion with high affinity and to the protein portion with low affinity (Maeda et al., 1999
).
The present investigation focuses on the structure of glycosaminoglycans involved in strong binding to MK. Using heparin and its partially desulfated derivatives as model compounds, we conclude that all three sulfate groups of heparin-disaccharide units (6-O, 2-O, and N-sulfates) are required for full interaction with MK, suggesting that the trisulfated unit in heparan sulfate is involved in MK binding (Kaneda et al., 1996a). Furthermore, CS-E, which has a disaccharide unit with 4,6-disulfated N-acetylgalactosamine, has been shown to bind to MK with an affinity as strong as heparin (Ueoka et al., 2000
). However, little information is available about the structure of glycosaminoglycans actually present in MK-responding cells (Ueoka et al., 2000
; Zou et al., 2000
). In addition, the presence of large amounts of 3-O-sulfation in naturally occurring CS-E (Kinoshita et al., 1997
) hinders the precise identification of the structure required for strong binding of CS-E to MK.
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Results |
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Each CS fraction from mouse embryos was digested with chondroitinase ABC, and the released unsaturated disaccharides were analyzed by high-performance liquid chromatography (HPLC) (Figure 2AC). Only Di-4S derived from CS-A structure and
Di-diSE (arrows) derived from CS-E structure were detected in significant amounts. Although
Di-diSB is also eluted in the fraction, the disulfated form was confirmed to be mostly
Di-diSE because more than 90% of the radioactivity was susceptible to chondro-6-sulfatase (Figure 2D). The percent of
Di-diSE in the total unsaturated disaccharides was calculated after correcting for the presence of a small amount of
Di-diSB; the value progressively increased in the more strongly binding fraction and was 16.7% in the 0.3 M NaCl fraction, 27.3% in the 0.5 M NaCl fraction, and 32.3% in the 0.7 M fraction.
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To clarify the point, we utilized artificial CS-E formed by sulfation of C4S from rat cartilage using purified N-acetylgalactosamine-4-sulfate 6-O-sulfotransferase (4S6ST). The artificial CS-E behaved similarly to authentic CS-E on MK-Sepharose affinity chromatography (Figure 4A). The content of the E unit progressively increased in fractions eluted from the MK-Sepharose column with increasing concentration of NaCl; the value for the 0.7 M fraction was 57.2% (Table I, column A). When the artificial CS-E was digested with chondroitinase ACII and the products analyzed by Superdex 300 column chromatography, only 2% and 6% radioactivity were eluted in the tetrasaccharide and excluded volume, respectively, and rest of the radioactivity was in the disaccharide (Habuchi et al., 2002). The majority of the radioactivity in the void volume was slowly digested by ACII, suggesting that some of the oversulfated structure is more resistant to ACII. Structures involving 3-O-sulfated glucuronic acid (Kinoshita et al., 1997
) as well as L-iduronic acid are resistant to ACII. Therefore, 3-O-sulfated glucuronic acid is concluded to be present only in small amounts, if any, in the artificial CS. Thus, we concluded that a cluster of E units is required for strong binding to the MK column, and 3-O-sulfated glucuronic acid is not necessary for it.
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Disaccharide composition of MK-binding heparan sulfate from the brains of 13-day mouse embryos
[14C]-glucosamine-labeled glycosaminoglycans produced by the brain cells of 13-day mouse embryos were digested with chondroitinase ABC to remove CS and applied to an MK-Sepharose column; 89% of the bound radioactivity was eluted with 0.5 M NaCl and 11% with a higher NaCl concentration. Both glycosaminoglycans were digested with the mixture of heparitinase I, heparitinase II, and heparinase. HPLC analysis of the digest revealed arrays of the heparan sulfatederived unsaturated disaccharides (Figure 5). Comparing the disaccharide composition of the 0.5 M NaCleluted fraction and that of the strongly binding fraction, significant differences were found. In the strongly binding fraction, the trisulfated one, DiHS-triS (arrow, Figure 5B), was the most abundant sulfated product, and its content in the total unsaturated disaccharides was 17.2%. In case of the 0.5 Meluted fraction,
DiHS-NS (arrow, Figure 5A), was the most abundant sulfated product, and the
DiHS-triS content in the total unsaturated disaccharides was 11.7%. The identity of
DiHS-triS and that of a disulfated one,
DiHS-diS2, were also confirmed by gel filtration on a column of Bio-Gel P-4 (Figure 6).
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Discussion |
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The CS, which was synthesized by the embryonic brain and exhibited strong binding activity to MK, contained a DS domain, as shown by its susceptibility to chondroitinase B and partial resistance to ACII. This is consistent with our previous observation that chondroitinase B treatment of osteoblast-like cells (Qi et al., 2001) and macrophages (Hayashi et al., 2001
) abolishes the responsiveness to MK. CS in the MK-binding proteoglycan PG-M/ versican is also a hybrid-type chain with a DS domain (Zou et al., 2000
). However, it is unlikely that the DS domain is required for strong binding to MK or enhances the binding. Chondroitinase B treatment of CS-E formed on the backbone of rat cartilage C4S as well as CS-E from squid cartilage converted only a part of the CS to a weakly binding fraction (unpublished data). That the artificial E structure formed on DS also requires many E units for strong binding is consistent with this view. DS neither strongly inhibits the binding of MK to PTP
(Maeda et al., 1999
) nor inhibits the MK-induced neurite outgrowth of embryonic neurons (Kaneda et al., 1996b). Nevertheless, DS inhibits the migration of osteoblast-like cells (Qi et al., 2001
) or macrophages (Hayashi et al., 2001
). The DS domain may be required for interaction of the glycans with other molecules involved in cell migration.
We noticed that the E unit content in each strongly binding fraction varies significantly (Table I, Figure 2). We consider that a certain length of continuous E units is required for strong binding. This assumption is supported by the observation that the Di-6S content was inversely correlated with the binding capability to MK. However, the exact length of the continuous E units, which might be required for strong binding, was not clarified.
We also observed that commercial preparation of CS-D did not contain much strongly binding fraction. However, we cannot exclude the possibility that a dense cluster of D units might bind strongly to MK, because the commercial preparation contains considerable amounts of 6-monosulfated N-acetylgalactosamine residue.
Previously, CS-E unit was detected in the brains of embryonic rats (Ueoka et al., 2000) and PG-M/versican isolated from embryonic mouse brain (Zou et al., 2000
); however, the E unit content was only 23% in both cases. In the present study, we labeled the glycosaminoglycans by [14C]-glucosamine, so that the sensitivity of detection was increased and the disaccharide composition of glycosaminoglycans with the strongly binding fraction could be analyzed. We found that the E unit content was relatively high even in the weakly binding fraction. We believe that the labeled glycosaminoglycans reflect those in situ (because labeling was performed for 72 h) and not the dispersed cells, but pieces of the dissected brain were used for labeling. Recently, it has been reported that appican produced by cultured glioma cells contains 14.3% CS-E units (Tsuchida et al., 2001
). Thus, it is possible that in the embryonic brain, some class of proteoglycans carries clustered E unit. One of the obvious candidates is receptor-type tyrosine phosphatase
(Maeda et al., 1999
), but this point remains to be clarified.
We also characterized the composition of heparan sulfate chains in glycosaminoglycans with strong MK-binding activity. The trisulfated disaccharide unit, DiHS-triS, was the most abundant sulfated disaccharide released by heparitinases and heparinase. This finding extends the results obtained by previous studies; selective desulfation of any sulfate groups (2-O, 6-O, or N-) from heparin dramatically reduced the MK-binding activity of heparin (Kaneda et al., 1996a
). Considering these together, we reached the conclusion that the trisulfated structure on heparan sulfate chains also serves as strong binding site for MK in the responding cells.
Finally, recombinant 4S6ST produced CS-E, whose 4,6-disulfate content was extremely high. This is the first demonstration that this human enzyme is capable of forming an internally sulfated structure with high efficiency. Therefore, the enzyme is expected to be involved in the formation of the strong binding site for MK. Recently, oversulfated CS/DSs containing GlcAß1/IdoA1-3 GalNAc(4,6-O-disulfate) have been shown to interact with L- and P-selectins and chemokines (Kawashima et al., 2002
). Thus the enzyme might also be important for the formation of binding sites for chemokines and selectins.
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Materials and methods |
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Metabolic labeling of glycosaminoglycans
Fourteen brains of day-13 mouse embryos were excised and dissected with the aid of scissors in 9 ml Dulbecco's modified Eagle's medium containing 10 % fetal calf serum. The suspension was plated into a 10-cm tissue culture plate, and 15 µCi of [14C]-glucosamine was added per plate. After incubating for 72 h, the cells were collected and washed with phosphate buffered saline without Ca2+ and Mg2+[PBS(-)]. Twelve plates of the cultures were prepared.
Preparation of MK-binding CS
Radiolabeled embryonic brain cells were digested with 2 mg proteinase K in 2 ml PBS(-) containing 10 mM CaCl2 for 48 h at 37°C. The digest was boiled for 10 min, followed by centrifugation at 8000xg for 10 min. Then 0.1 U heparitinase I and II were added. The digestion was stopped by boiling for 2 min. After centrifuging at 8000xg for 10 min, the supernatant was applied to a Sephadex-50 column (1.4x90 cm) equilibrated in 50 mM Tris-HCl buffer, pH 7.5, and the CS-rich fraction eluted at the excluded volume was pooled. The fraction was applied to 1 ml MK-Sepharose column, and the MK-binding fraction was successively eluted with 10 ml 0.3 M, 0.5 M, and 1.5 M NaCl in 20 mM sodium phosphate, pH 7.2. For enzymatic digestion, the MK-binding CS was concentrated with Centricon YM-3 (Millipore, Bedford, MA) and diluted with 20 mM sodium phosphate, pH 7.2, to a final concentration of 0.15 M NaCl.
Preparation of MK-binding heparan sulfate
The [14C]-glucosamine-labeled MK-binding heparan sulfate was prepared as for the CS mentioned, except that the glycosaminoglycans were digested with 1 U chondroitinase ABC and the labeled heparan sulfate was eluted from the MK column by 10 ml each of 20 mM sodium phosphate buffer, pH 7. 2, with 0.5 M and 1.5 M NaCl.
Preparation of recombinant 4S6ST
To express the catalytic domain of human 4S6ST (Ohtake et al., 2001) as a recombinant secretory protein, two 4S6ST cDNA fragments were isolated from clone KIAA0598 (Verkoczy et al., 1998
), which was kindly given by Kazusa DNA Research Institute (Kisarazu, Chiba, Japan); one is 428 bp of BamHI/StuI fragment amplified by polymerase chain reaction with primers, 5'GGGATCCCCAAGAG-CTTCTGATCTCATCA3' and 5'GTGCGCCAGGTGG-CCCCAGAAGGCCT 3', and the other is 1222 bp of StuI/EcoRI fragment excised from KIAA0598. These cDNA fragments were ligated into pBluescript SKII() (Stratagene, CA) cleaved by SpeI/EcoRI in combination with the NheI/BamHI cDNA fragment encoding the melittin signal sequence, (His)6 tag, and enterokinase-susceptible sequence, all of which were isolated from the MK transfer vector (Asai et al., 1997
). Finally, the XbaI/EcoRV fragment excised from the resulting plasmid was inserted into the XbaI/SmaI-cleaved baculovirus transfer vector pVL1393 (Pharmingen, CA). The production of the recombinant 4S6ST protein in Sf-21 cells was performed as described previously (Kaneda et al., 1996b
).
The recombinant enzyme secreted into the conditioned medium (600 ml) was applied to a 1-ml column of heparin Sepharose CL-6B (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 50 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl. After washing with the same buffer, the enzyme solution was eluted with 6 ml of the buffer containing 0.5 M NaCl. For further purification, the eluate was applied to 1 ml HisTrap chelating HP column (Amersham Pharmacia Biotech), washed with 40 mM imidazole, and eluted with 5 ml 0.1 M and 0.2 M imidazole according to the manufacturer's instructions. The recombinant enzyme eluted with 0.2 M imidazole gave a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The purified enzyme was concentrated and desalted using Centricon YM-10 (Millipore). From 600 ml of the medium, 150 µg of the enzyme protein was obtained. The specific activity of the recombinant enzyme was 26.7 mU per mg protein.
Artificial CS formed on the backbone of [35S]-labeled epiphyseal cartilage from newborn rat
The details of the procedure will be published elsewhere (Habuchi et al., 2002). Epiphyseal cartilage was dissected from newborn rats and incubated with [35S]O4. The labeled cartilage was digested with actinase, and proteins were removed by precipitation with 5% trichloroacetic acid. The [35S]O4-labeled CS (25 nmole as galactosamine, about 3x105 cpm) was incubated with unlabeled phosphoadenyl 5'-phosphosulfate and 180 ng purified 4S6ST as described elsewhere (Ito et al., 2000
). The product contained 43%
Di-diSE.
Artificial CS formed on the backbone ofcommercial CSs
CSs or DS (250 µg) was incubated with 74 µg of the recombinant 4S6ST and 500 µg phosphoadenyl 5'-phosphosulfate in 1 ml 20 mM sodium phosphate buffer, pH 7.4, containing 10 mM CaCl2, 20 mM reduced glutathione, and 50 mM imidazole at 37°C for 12 h. The product was then fractionated by MK-Sepharose affinity chromatography.
Analysis of disaccharide composition of CSs
Samples were digested with 0.1 U chondroitinase ABC in 0.1 ml 20 mM sodium phosphate buffer, pH 8.0, containing 50 mM acetate at 37°C for 6 h. Samples before or after chondro-6-sulfatase treatment (0.1 U at 37°C for 6 h) were then analyzed by HPLC (Partisil-10 SAX, 4.6x250 mm, Whatman) as described by Fukuta et al. (1995).
Analysis of disaccharide composition of heparan sulfate
Heparinase, heparitinase I, and heparitinase II digestion was performed in 20 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl and 2 mM CaCl2, using 0.15 U/ml for each enzyme, at 37°C for 20 h. All three enzymes were used to depolymerize heparan sulfate completely. Disaccharides released from heparan sulfate were analyzed on a CarboPac PA-1 column (4x250 mm, Dionex), eluted with a LiCl gradient as described previously (Maccarana et al., 1996). The collection was started at 3.6 min after injection and 0.33 ml/fraction was collected at a flow rate of 0.8 ml/min. The radioactivity of each fraction was determined by liquid scintillation counting.
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Acknowledgements |
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: tmurama{at}med.nagoya-u.ac.jp
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Abbreviations |
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References |
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