Glycosaminoglycan structures required for strong binding to midkine, a heparin-binding growth factor

Peng Zou2, Kun Zou2, Hisako Muramatsu2, Keiko Ichihara-Tanaka2, Osami Habuchi3, Shiori Ohtake3, Shinya Ikematsu4, Sadatoshi Sakuma4 and Takashi Muramatsu12

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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 Results
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Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, {Delta}Di-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, {Delta}DiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.

Key words: chondroitin sulfate / heparan sulfate / sulfotransferase


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 Abstract
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Recognition of glycosaminoglycans by growth factors is essential for the action of a class of growth factors, whose typical examples are fibroblast growth factors (FGFs) (Bernfield et al., 1999Go; Park et al., 2000Go; Pellegrini et al., 2000Go; Plotnikov et al., 1999Go; Ruoslahti and Yamaguchi, 1991Go; Yayon et al., 1991Go). FGF, FGF receptor, and heparan sulfate form a trimolecular complex to deliver the FGF signal into cells (Pellegrini et al., 2000Go; Plotnikov et al., 1999Go).

Midkine (MK) is a heparin-binding growth factor structurally unrelated to FGFs (Iwasaki et al., 1997Go; Kadomatsu et al., 1988Go; Muramatsu, 2002Go; Muramatsu et al., 1993Go; Owada et al., 1999Go; Tomomura et al., 1990Go) and together with heparin-binding growth-associated molecule (HB-GAM; also called pleiotrophin) (Li et al., 1990Go; Merenmies and Rauvala, 1990Go; Rauvala, 1989Go) forms a new family of heparin-binding growth factors. MK promotes neurite outgrowth (Muramatsu et al., 1993Go; Muramatsu and Muramatsu, 1991Go), survival (Owada et al., 1999Go), growth (Muramatsu and Muramatsu, 1991; Takei et al., 2001Go), and migration (Horiba et al., 2000Go; Maeda et al., 1999Go; Qi et al., 2001Go; Takada et al., 1997Go) of various cells and is involved in tumor growth (Takei et al., 2001Go), vascular diseases (Horiba et al., 2000Go), and nephritis induced by ischemia (Sato et al., 2001Go).

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., 1996bGo; Kinnunen et al., 1996Go). The C-terminal half of MK, which has a conformation-dependent heparin binding site, can promote neurite outgrowth (Muramatsu et al., 1994Go). 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., 1994Go). Recently, a chondroitin sulfate (CS) proteoglycan, receptor-type protein tyrosine phosphatase {zeta}, has been found as a receptor of MK (Maeda et al., 1999Go; Qi et al., 2001Go) and HB-GAM (Maeda and Noda, 1998Go) in migration of embryonic neurons (Maeda et al., 1999Go; Maeda and Noda, 1998Go) and osteoblasts (Qi et al., 2001Go). MK binds to the CS portion with high affinity and to the protein portion with low affinity (Maeda et al., 1999Go).

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., 1996aGo). 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., 2000Go). However, little information is available about the structure of glycosaminoglycans actually present in MK-responding cells (Ueoka et al., 2000Go; Zou et al., 2000Go). In addition, the presence of large amounts of 3-O-sulfation in naturally occurring CS-E (Kinoshita et al., 1997Go) hinders the precise identification of the structure required for strong binding of CS-E to MK.


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 Abstract
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 Results
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 References
 
Disaccharide composition of MK-binding CS from the brain of 13-day mouse embryos
[C14]-Glucosamine-labeled glycosaminoglycans from the brains of 13-day mouse embryos were digested with heparitinase to remove heparan sulfate and applied to a MK-Sepharose column. About 4% of radioactivity were bound to the column, even after elution with 0.5 M NaCl, and were eluted by 0.7 M NaCl (Figure 1Aa). On rechromatography, most of the radioactivity eluted by 0.7 M NaCl behaved identically (Figure 1Ab, solid line).



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Fig. 1. MK-Sepharose affinity column chromatography of glycosaminoglycans. Various specimens of glycosaminoglycans in 1 ml 20 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl (200 nmol in uronic acids in the case of unlabeled ones) were applied to a column of MK-Sepharose (0.2 ml) equilibrated with the buffer containing 0.15 M NaCl. The unabsorbed fraction was reapplied to the column, and the procedure was repeated once. After washing with 2 ml of the buffer with 0.15 M NaCl, glycosaminoglycans were eluted with 2 ml of the buffer containing 0.3, 0.5, 0.7, 0.9, and 2 M NaCl. No glycosaminoglycans were eluted by the buffer with 2 M NaCl. (A) a, [14C]-Glucosamine-labeled CS-rich glycosaminoglycans synthesized by the dissected brain from day-13 mouse embryos; b, the strongly binding fraction eluted by 0.7 M NaCl in a was rechromatographed either before (solid line) or after (dashed line) chondroitinase B digestion. Fractions of 0.2 ml were collected, and each fraction was assayed for radioactivity. (B) Commercially available glycosaminoglycans. Fractions of 1 ml were collected, and each fraction was assayed by carbazole reaction (Bitter and Muir, 1962Go). The combined value of uronic acid in each NaCl-concentration fraction is shown.

 
We examined the behavior of naturally occurring glycosaminoglycans on MK-affinity chromatography (Figure 1B). All of the glycosaminoglycans bound to the column in the presence of 0.15 M NaCl. The majority of CS-E was bound to the column after elution with 0.5 M NaCl and was eluted by 0.7 M NaCl and a small amount by 0.9 M NaCl (CS-E). However, only small amounts of chondroitin 6-sulfate (C6S), CS-D, and dermatan sulfate (DS) and no chondroitin 4-sulfate (C4S) were recovered in the 0.7 M fraction. None of these CSs were in the 0.9 M fraction. Heparin behaved similarly to CS-E. The amount of material eluted by a NaCl concentration of 0.7 M and higher correlated with the MK-binding activity determined by surface plasmon resonance and the ability to inhibit neurite outgrowth of embryonic neurons (Kaneda et al., 1996bGo; Ueoka et al., 2000Go). Thus we regarded those retained in the column by washing with 0.5 M NaCl and eluted from it by a higher NaCl concentration to be the strongly binding fraction.

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 2A–C). Only {Delta}Di-4S derived from CS-A structure and {Delta}Di-diSE (arrows) derived from CS-E structure were detected in significant amounts. Although {Delta}Di-diSB is also eluted in the fraction, the disulfated form was confirmed to be mostly {Delta}Di-diSE because more than 90% of the radioactivity was susceptible to chondro-6-sulfatase (Figure 2D). The percent of {Delta}Di-diSE in the total unsaturated disaccharides was calculated after correcting for the presence of a small amount of {Delta}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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Fig. 2. Disaccharide analysis of [14C]-glucosamine-labeled CS that was synthesized by the brain and separated by MK-Sepharose column chromatography. After digestion with chondroitinase ABC, the resulting disaccharide was separated by HPLC. Arrows indicate the elution position of standards; 1, {Delta}Di-0S; 2, {Delta}Di-6S; 3, {Delta}Di-4S; 4, {Delta}Di-diSD; 5, {Delta}Di-diSB; 6 and arrows, {Delta}Di-diSE; 7, {Delta}Di-triS. (A) Fraction eluted by 0.3 M NaCl; (B) fraction eluted by 0.5 M NaCl; (C) fraction eluted by 1.5 M NaCl; (D) the 0.5 M NaCl fraction digested with chondroitinase ABC and chondro-6-sulfatase.

 
When the strongly binding fraction eluted by 0.7 M NaCl was digested with chondroitinase B, the affinity became weak, indicating that a DS domain is present in the CS (Figure 1Ab, broken line). This point was confirmed by digestion with chondroitinase ABC and ACII; the former enzyme completely depolymerized the CS preparation (Figure 3A), whereas ACII, which does not act on DS domain, only partially depolymerized it (Figure 3B).



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Fig. 3. Sephadex G-50 column chromatography of [14C]-glucosamine-labeled CS eluted from the MK column by 0.7 M NaCl. The strongly binding fraction (see Figure 1a) was digested with 0.1 U chondroitinase ABC (A) or ACII (B) in 0.2 ml 20 mM sodium phosphate buffer, pH 7.2, and was applied to a column of Sephadex G-50, fine (1x54 cm), which was equilibrated and eluted with 0.05 M NH4HCO3. Fractions of 0.6 ml were collected. The position of the elution position of blue dextran (Vo) and lactose (Lac) are shown.

 
MK-binding capability of artificially formed CS-E
CS-E from squid cartilage contains significant amounts of 3-O sulfated group linked to glucuronic acid (Kinoshita et al., 1997Go). Because the sulfate group is released on chondroitinase digestion, extensive biochemical analysis is required for its identification; we could not exclude the possibility that such a structure is present in CS produced by the embryonic brain cells and contributes to MK binding.

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., 2002Go). 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., 1997Go) 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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Fig. 4. MK-Sepharose affinity chromatography of artificial CSs, whichwas formed using purified (A) or recombinant (BD) 4S6ST. Experiments were performed as described in Figure 1 except that the amounts of the applied glycosaminoglycans were 500 nmoles as uronic acids, leading to unabsorption of a part of the material. (A) Oversulfated C4S formed on rat cartilage one; (B) oversulfated C4S formed on whale cartilage one; (C) oversulfated C4S formed on sturgeon notochord one; (D) oversulfated DS.

 

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Table I. Disaccharide composition of artificially formed chondroitin sulfates separated by MK-Sepharose affinity chromatography with increasing concentrations of NaCl

 
We also transferred sulfate to the 6-position of 4-sulfated N-acetylgalactosamine in C4S from whale cartilage and from sturgeon notochord and in DS. For these experiments, we used human 4S6ST produced in the baculovirus system because the recombinant enzyme could be obtained in large amounts. Only a small fraction of whale CS was converted to the strongly binding fraction after the sulfotransferase action (Figure 4B). C4S from the sturgeon notochord was moderately converted to the strongly binding fraction (Figure 4C), and DS was effectively converted to it. Analysis of the disaccharide composition again revealed that a greater content of the E unit was present in the strongly binding fraction (Table I). The trisulfated unit, {Delta}Di-triS, was present in small amounts in the fractions from DS. The content of {Delta}Di-6S inversely correlated with the binding capability. Furthermore, a lower content of {Delta}Di-diSE was sufficient to form the strongly binding fraction when the {Delta}Di-6S content was low (Table I). Because even the 0.5 M NaCl fraction of the DS-derived artificial E structure was mainly composed of the E unit (Table I, column D), DS domain did not enhance the binding to MK Sepharose.

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 sulfate–derived unsaturated disaccharides (Figure 5). Comparing the disaccharide composition of the 0.5 M NaCl–eluted fraction and that of the strongly binding fraction, significant differences were found. In the strongly binding fraction, the trisulfated one, {Delta}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 M–eluted fraction, {Delta}DiHS-NS (arrow, Figure 5A), was the most abundant sulfated product, and the {Delta}DiHS-triS content in the total unsaturated disaccharides was 11.7%. The identity of {Delta}DiHS-triS and that of a disulfated one, {Delta}DiHS-diS2, were also confirmed by gel filtration on a column of Bio-Gel P-4 (Figure 6).



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Fig. 5. Analysis of the disaccharides released from [14C]-glucosamine-labeled heparan sulfate, which was synthesized by brain cells of day-13 mouse embryos and separated by MK-Sepharose affinity chromatography by increasing NaCl concentrations. MK-binding heparan sulfate was completely cleaved with a mixture of heparinase, heparitinase I, and heparitinase II and separated by HPLC on a column of CarboPac PA-1. (A) 0.5 M NaCl eluate; (B) 1.5 M NaCl eluate. Elution positions of standards are indicated by arrows: 1, {Delta}DiHS-0S; 2 and an arrow in A, {Delta}DiHS-NS; 3, {Delta}DiHS-6S; 4, {Delta}DiHS-2S; 5, {Delta}DiHS-diS1; 6, {Delta}DiHS-diS2;7 and an arrow in B, {Delta}DiHS-triS.

 


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Fig. 6. Identification of disaccharides separated by HPLC by gel filtration on a column of Bio-gel P-4. [14C]-Labeled disaccharides released from heparan sulfate with strong MK-binding activity (see Figure 5B) were mixed with 100 µg each of authentic standards and applied to a column of Bio-gel P-4 (0.8x115 cm), which was equilibrated and eluted with 0.5 M NH4HCO3, and 0.64-ml fractions were collected. The materials eluted in the position of {Delta}DiHS-diS2 (open circles) and {Delta}DiHS-triS (closed circles) were analyzed. 1–5, Dextran oligomers (the number corresponds to polymerization units: n = 1, glucose); 6, {Delta}DiHS-diS2; 7, {Delta}DiHS-triS; 8, blue dextran.

 

    Discussion
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The present investigation clearly revealed that for strong binding of CSs to MK, multivalent E units are required and sufficient. First, we analyzed [14C]-glucosamine-labeled CS produced by the brains of day-13 mouse embryos in which MK is abundantly expressed and is expected to work locally. The CS, which bound to MK strongly, contained about 32% of the E unit, which was recovered as {Delta}Di-diSE after chondroitinase ABC digestion. Second, artificial CS-E formed on the backbone of C4S from rat cartilage or from sturgeon notochord contained a significant fraction of the strongly binding fraction. The result excluded the possibility that 3-O-sulfated glucuronic acid is essential for the strong binding. It is noteworthy that the binding capability of artificial CS-E, which has two sulfate residues per a disaccharide unit, was similar to that of heparin, which has three sulfate residues per a disaccharide unit. Therefore, MK–glycosaminoglycan interactions depend on the specific structure of glycosaminoglycans and are not simple electrostatic interactions.

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., 2001Go) and macrophages (Hayashi et al., 2001Go) 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., 2000Go). 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{zeta} (Maeda et al., 1999Go) 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., 2001Go) or macrophages (Hayashi et al., 2001Go). 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 {Delta}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., 2000Go) and PG-M/versican isolated from embryonic mouse brain (Zou et al., 2000Go); however, the E unit content was only 2–3% 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., 2001Go). 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 {zeta} (Maeda et al., 1999Go), 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, {Delta}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., 1996aGo). 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/IdoA{alpha}1-3 GalNAc(4,6-O-disulfate) have been shown to interact with L- and P-selectins and chemokines (Kawashima et al., 2002Go). Thus the enzyme might also be important for the formation of binding sites for chemokines and selectins.


    Materials and methods
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Materials
Recombinant human MK was expressed in yeast and purified as described elsewhere (Murasugi and Tohma-Aiba, 2001Go; Ikematsu et al., 2000Go). An MK-Sepharose column was prepared as described by coupling 5 mg MK per ml of resin (Muramatsu et al., 2000Go). D-[1-14C] glucosamine hydrochloride (specific activity 45–60 mCi/mmol) was obtained from NEN Life Science Products (Boston, MA). Proteinase K was obtained from Wako Pure Chemicals (Osaka, Japan). Heparinase, heparitinase I, heparitinase II, chondroitinase ABC, chondroitinase B, chondroitinase AC II , chondro-6-sulfatase, C4S from whale cartilage and from sturgeon notochord, C6S from shark cartilage, DS from pig skin, CS-D from shark cartilage, CS-E from squid cartilage, and {Delta}Di-0S, {Delta}Di-4S, {Delta}Di-6S, {Delta}Di-SD, {Delta}Di-SE, {Delta}Di-tris, {Delta}DiHS-0S, {Delta}DiHS-NS, {Delta}DiHS-6S, {Delta}DiHS-diS1, {Delta}DiHS-diS2, and {Delta}DiHS-triS were purchased from Seikagaku Kogyo (Tokyo). 4S6ST was purified as described elsewhere (Ito and Habuchi, 2000Go).

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., 2001Go) as a recombinant secretory protein, two 4S6ST cDNA fragments were isolated from clone KIAA0598 (Verkoczy et al., 1998Go), 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., 1997Go). 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., 1996bGo).

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 sulfate–polyacrylamide 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., 2002Go). 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., 2000Go). The product contained 43% {Delta}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)Go.

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., 1996Go). 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.


    Acknowledgements
 
This work was supported by Scientific Research Grants-in-aid 10CE2006 and 14082202 from the Ministry of Education, Science, Sports, Culture and Technology of Japan. We thank Tomoko Adachi and Hitomi Inoue for their secretarial assistance.


    Footnotes

1 To whom correspondence should be addressed; e-mail: tmurama{at}med.nagoya-u.ac.jp Back


    Abbreviations
 
4S6ST, N-acetylgalactosamine4-sulfate 6-O-sulfotransferase; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; FGF, fibroblast growth factor; HB-GAM, heparin-binding growth-associated molecule; HPLC, high-performance liquid chromatography; MK, midkine; PBS(-), Dulbecco's phosphate buffered saline without Ca2+ and Mg2+.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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