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Originally published In Press as doi:10.1074/jbc.M609296200 on December 4, 2006

J. Biol. Chem., Vol. 282, Issue 5, 2956-2966, February 2, 2007
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Neuritogenic Activity of Chondroitin/Dermatan Sulfate Hybrid Chains of Embryonic Pig Brain and Their Mimicry from Shark Liver

INVOLVEMENT OF THE PLEIOTROPHIN AND HEPATOCYTE GROWTH FACTOR SIGNALING PATHWAYS*

Fuchuan Li{ddagger}§1, Ajaya Kumar Shetty{ddagger}§, and Kazuyuki Sugahara{ddagger}§2

From the {ddagger}Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 and the §Department of Biochemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan

Received for publication, October 2, 2006 , and in revised form, November 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence suggests the involvement of chondroitin sulfate (CS) and dermatan sulfate (DS) hybrid chains in the brain's development and critical roles for oversulfated disaccharides and IdoUA residues in the growth factor-binding and neuritogenic activities of these chains. In the pursuit of sources of CS/DS with unique structures, neuritogenic activity, and therapeutic potential, two novel CS/DS preparations were isolated from shark liver by anion exchange chromatography. The major (80%) low sulfated and minor (20%) highly sulfated fractions had an average molecular mass of 3.8–38.9 and 75.7 kDa, respectively. Digestion with various chondroitinases (CSases) revealed a large panel of disaccharides with either GlcUA or IdoUA scattered along the polysaccharide chains in both of the fractions. The higher Mr fraction, richer in IdoUA(2-O-sulfate){alpha}1–3GalNAc(4-O-sulfate) and GlcUAbeta/IdoUA{alpha}1–3GalNAc(4,6-O-disulfate) units, exerted greater neurite outgrowth-promoting (NOP) activity and better promoted the binding of various heparin-binding growth factors, including pleiotrophin (PTN), midkine, recombinant human heparin-binding epidermal growth factor-like growth factor, VEGF165, fibroblast growth factor-2, fibroblast growth factor-7, and hepatocyte growth factor (HGF). These activities were largely abolished by digestion with CSase ABC or B but only moderately affected by a mixture of CSases AC-I and AC-II. In addition, the NOP activity of the larger fraction was markedly reduced by desulfation with alkali, suggesting a role for the 2-O-sulfate of IdoUA(2-O-sulfate){alpha}1–3GalNAc(4-O-sulfate). The NOP activity of the higher molecular weight fraction and that of the embryonic pig brain-derived CS/DS fraction were also sup pressed to a large extent by antibodies against HGF, PTN, and their individual receptors cMet and anaplastic lymphoma kinase, revealing the involvement of the HGF and PTN signaling pathways in the activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosaminoglycan (GAG)3 side chains of proteoglycans, namely chondroitin sulfate (CS)/dermatan sulfate (DS) are widely expressed at the cell surface and in extracellular matrices (1, 2). CS and DS chains consist of repeating disaccharide units of -GlcUA-GalNAc- and -IdoUA-GalNAc- with various sulfation patterns, respectively, and often exist as CS/DS co-polymeric structures (3, 4). There is evidence to suggest that CS/DS plays crucial roles in biological events, such as the development of the central nervous system (5–8), wound repair (9, 10), viral attachment (11–13), growth factor signaling (14, 15), morphogenesis (16), and cytokinesis (17–19). Among these events, the development of the central nervous system, involving neuronal adhesion, migration, and neurite formation, has recently attracted attention in terms of the functions of CS/DS (4, 20).

The disaccharide composition of CS/DS chains in the brain shows developmental changes (21–23), suggesting that these chains differing in the ratio of GlcUA and IdoUA and in the sulfation profile may exert distinct functions during the brain's development. The proportion of oversulfated disaccharides and the presence of IdoUA are two crucial factors for the neurite outgrowth-promoting (NOP) activity of CS/DS chains (24–28), and the CS/DS chains expressed at the surface of neuronal cells or immobilized on the matrices exert NOP activity and bind various heparin-binding growth factors in vitro.

Our laboratory has recently shown that endogenous pleiotrophin (PTN) (29) or heparin-binding growth-associated molecule (30), which is a neuritogenic growth factor (31), is recruited by the CS/DS chains from pig embryonic brain (E-CS/DS) and mediates the NOP activity of the CS/DS chains (32). Five octasaccharide sequences with at least one GlcUA(2S)beta1–3GalNAc(6S) (D) or IdoUA(2S){alpha}1–3GalNAc(6S) (iD) disaccharide, where 2S and 6S stand for 2-O- and 6-O-sulfate, respectively, have been isolated from a subfraction with low affinity for PTN after enzymatic fragmentation of the polysaccharides, followed by affinity chromatography using a PTN-immobilized column (33).

To search for sources of CS/DS chains with therapeutic potential, CS/DS hybrid chains were purified from shark liver and found to have a unique structure and strong NOP activity. Further study revealed the molecular mechanism of the NOP activity to involve the signaling pathway of not only PTN but also hepatocyte growth factor (HGF).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Livers of Prionace glauca (blue shark) were provided by Maruha Group Inc., Central Research Institute (Tsukuba-City, Japan). Pregnant ddY mice were purchased from SLC Inc. (Shizuoka, Japan). CS-A from whale cartilage; CS-B from porcine skin; CS-C and CS-D from shark cartilage; CS-E from squid cartilage; and standard unsaturated disaccharides, chondroitinases (CSases) ABC (EC 4.2.2.4 [EC] ), AC-I (EC 4.2.2.5 [EC] ), and AC-II (EC 4.2.2.5 [EC] ), and hyaluronidase SH (EC 4.2.2.1 [EC] ) from Streptomyces hyalurolyticus were purchased from Seikagaku Corp. (Tokyo, Japan). CSase B was obtained from IBEX Technologies (Montreal, Canada). Embryonic pig brain-derived CS/DS (E-CS/DS) and its high affinity fraction (E-CS/DS-H) were prepared as described previously (32). Recombinant human (rh) pleiotrophin (PTN) expressed in E. coli and rh-vascular endothelial growth factor-165 (VEGF165) expressed in insect cells were from RELIA Tech GmbH (Braunschweig, Germany). rh-Midkine (MK) expressed in Escherichia coli and rh-fibroblast growth factor (FGF)-1 (or acidic FGF) expressed in E. coli were from PeproTech EC Ltd. (London, UK). rh-FGF-2 (or basic FGF) expressed in E. coli was from Genzyme TECHNE (Minneapolis, MN). rh-Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and rh-hepatocyte growth factor/scatter factor (HGF/SF) expressed in Sf21 insect cells, rh-keratinocyte growth factor (KGF/FGF-7) expressed in E. coli, and anti-mouse HGF receptor IgG were obtained from R&D Systems (Minneapolis, MN). 1,9-Dimethylmethylene blue was from Aldrich. Polyclonal goat anti-rh-PTN IgG and polyclonal goat anti-rh-MK IgG were obtained from Genzyme/Techne (Cambridge, MA). Anti-FGF-2/basic FGF, clone bFM-1, was from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (New York, NY). Polyclonal goat IgG against mouse anaplastic lymphoma kinase (ALK T-18), was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-rat HGF rabbit IgG was provided by Prof. Toshikazu Nakamura (Osaka University, Osaka, Japan). Purified serum IgG from mouse, goat, and rabbit were obtained from Sigma. Actinase E was from Kaken Pharmaceutical Co. (Tokyo, Japan). All other chemicals and reagents were of the highest quality available.

Extraction of SL-CS/DS—Livers of blue shark (P. glauca), were dehydrated and delipidated by extraction with acetone, air-dried, and used for extraction of GAGs essentially as described previously (28) with some modifications. Briefly, 65 g of the acetone powder, corresponding to 163 g of the wet tissue, was treated with actinase E, followed by 5% trichloroacetic acid to precipitate residual proteins and peptides and with ether to extract trichloroacetic acid. To extract GAGs exhaustively, the precipitate obtained with trichloroacetic acid was treated with 0.5 M NaOH at 4 °C for 20 h and then neutralized with 1 M acetic acid before being precipitated with trichloroacetic acid, extracted with ether, and combined with the GAG extract obtained by actinase digestion. A crude GAG fraction was recovered from the combined extract by precipitation with 80% ethanol containing 5% sodium acetate at 4 °C overnight. The yield was 9 g, containing 416 mg of GAG based on the carbazole reaction.

Purification of SL-CS/DS—The crude GAG fraction (1.5 g) was loaded on a DEAE-Sephadex column (15 x 300 mm) pre-equilibrated with 0.3 M phosphate buffer, pH 6.0, containing 0.2 M NaCl. After the column was washed with the equilibration buffer, GAGs were eluted with the same buffer containing 2.0 M NaCl, dialyzed against water, and concentrated to dryness (the yield was 25 mg). This sample was subjected to a nitrous acid treatment (pH 1.5) to remove heparin/HS as described previously (34), and the resultant HS fragments were removed by gel filtration on a Superdex® 75 column (10 x 300 mm; Amersham Biosciences) eluted with 0.2 M NH4HCO3 at a flow rate of 0.4 ml/min. The elution was monitored by absorption at 210 nm. The fraction eluted in the void volume was pooled and freeze-dried repeatedly by reconstituting in water to remove NH4HCO3. This CS/DS preparation was fractionated by anion exchange chromatography on an Accell QMA Plus cartridge (Waters, Milford, MA) and eluted stepwise with 0.3 M phosphate buffers (pH 6.0) containing 0.2, 1.0, 1.5, and 2.0 M NaCl. The fractions obtained by elution with 1.0 and 1.5 M NaCl, referred to as SL-CS/DS (1.0 M) and SL-CS/DS (1.5 M), respectively, were desalted using a PD-10 column (Amersham Biosciences), and each fraction was analyzed by the carbazole reaction for the amount of CS/DS (35). Finally, the SL-CS/DS preparations were passed through a Sep-PakC18 cartridge (Waters) to remove peptides.

Determination of the Disaccharide Composition and Molecular Mass—An aliquot (1 µg as GAG) of SL-CS/DS (1.0 M) or (1.5 M) was subjected to digestion with CSase ABC, a mixture of CSases AC-I and AC-II, or CSase B. Each digest was labeled with 2-aminobenzamide (2AB) and subjected to anion exchange HPLC on an amine-bound silica PA-03 column (YMC-Pack PA, Kyoto, Japan) as described previously (28). To determine the molecular mass, an aliquot (5 µg as GlcUA) of SL-CS/DS (1.0 and 1.5 M fractions) was chromatographed by gel filtration on a SuperdexTM 200 column (10 x 300 mm; Amersham Biosciences), which had been calibrated using a series of size-defined commercial polysaccharides (36). The sample was eluted with 0.2 M ammonium bicarbonate at a flow rate of 0.3 ml/min for 90 min. Fractions were collected at 3-min intervals, freeze-dried, and dissolved in 100 µl of water. An aliquot was utilized for estimating the total amount of GAGs using 1,9-dimethylmethylene blue according to the method of Chandrasekhar et al. (37), except that the absorbance was recorded at 525 nm.

Interaction Analysis—Inhibition of PTN binding to a PTN high affinity fraction derived from embryonic pig brain (E-CS/DS-H) was examined using a BIAcore J system (BIAcore AB, Uppsala, Sweden). E-CS/DS-H was immobilized on a sensor chip as previously described (32). PTN (100 ng) was mixed with 0.5 µg of each tested GAG preparation (SL-CS/DS fractions or commercial CS/DS preparations) and incubated for 15 min at room temperature prior to injection onto the surface of an E-CS/DS-H-immobilized sensor chip. Results are expressed as relative percentages of inhibition based on the binding of PTN to E-CS/DS-H in the absence of inhibitor as 100%. To examine the interaction with various growth factors, SL-CS/DS (1.5 M) was immobilized on a sensor chip as reported earlier (38). For kinetic analysis, various concentrations of growth factors were injected onto the surface of this sensor chip in the running buffer (HBS-EP, pH 7.4, BIAcore AB) with a medium flow rate (30 µl/min) as per the manufacturer's protocol. Each growth factor was allowed to interact with SL-CS/DS (1.5 M) for 2 min each for association and for dissociation, after which the sensor chip was regenerated by injecting 1 M NaCl for 2 min before each injection. The kinetic parameters were evaluated with BIAevaluation 3.1 software (BIAcore AB) using a 1:1 binding model with mass transfer. To investigate the structural characteristics of the putative functional epitopes of SL-CS/DS (1.5 M) for the binding to various growth factors, an aliquot (10 µgas GAG) was digested with 10 mIU each of CSase ABC, a mixture of CSases AC-I and AC-II, or CSase B, and a 2-µg aliquot of each digest was used for the inhibition analysis as described above except that the SL-CS/DS (1.5 M)-immobilized sensor chip was used here.

Preparation of Partially Desulfated CS/DS Chains—The alkali treatment, which removes 2-O-sulfate from the IdoUA of heparin (39), was applied to the SL-CS/DS (1.5 M) fraction. SL-CS/DS (1.5 M) (20 µg as GAG) was dissolved in 20 µl of 0.1 M NaOH, frozen at –20 °C for 2 h, neutralized with 0.5 M HCl to pH 7.0, desalted by gel filtration on a PD-10 column, and freeze-dried.

Assays for NOP Activity—Cultures of hippocampal neurons were established from E16 mice as previously described (25). Briefly, 2 µg/well of a CS/DS preparation was individually coated onto coverslips precoated with poly-DL-ornithine (P-ORN) (Sigma) at 4 °C overnight. The hippocampal neuronal cells were freshly isolated from E16 mouse embryos, suspended in Eagle's minimum essential medium containing N2 supplements, seeded on coverslips at a density of 10,000 cells/cm2, and allowed to grow in a humidified atmosphere for 24 h at 37 °C with 5% CO2.

For the neutralization assay using antibodies, polyclonal anti-PTN antibody (10 µg/ml), polyclonal anti-MK antibody (10 µg/ml), monoclonal anti-bFGF antibody (10 µg/ml), or polyclonal anti-HGF antibody (3 µg/ml) was added to the medium 2 h after the seeding of the cells. After incubation overnight, the cells were fixed using 4% (w/v) paraformaldehyde for 30 min at room temperature, and the neurites were visualized by immunochemical staining using anti-microtubule-associated protein-2 (Lieco Technologies Inc., St. Louis, MO) (40) and anti-neurofilament (Sigma) (41). The antibodies were then detected using a Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA) with 3,3'-diaminobenzidine as a chromogen. The immunostained cells on each coverslip were scanned and digitized with a x20 objective lens on an optical microscope (BH-2; Olympus, Tokyo, Japan) equipped with a digital camera (HC-300Z/OL; Olympus). 100 clearly isolated cells with at least one neurite longer than the cell body were chosen at random to determine the length of the longest neurite using a morphological analysis software (Mac SCOPE; Mitani Corp., Tokyo, Japan). At least three independent experiments per parameter or condition were carried out.

Analysis of Distribution of CS and DS Domains in the SL-CS/DS (1.5 M) Chains—SL-CS/DS (1.5 M)(1 µg as GAG) was digested with CSase ABC, a mixture of CSases AC-I and AC-II, or CSase B and labeled with 2AB as described above. Each digest was dissolved in 200 µl of 0.2 M NH4HCO3 and analyzed by gel filtration chromatography on a SuperdexTM Peptide HR column (10 x 300 mm; Amersham Biosciences), which was eluted with 0.2 M NH4HCO3 at a flow rate of 0.4 ml/min, being monitored with a RF-10AXL Shimadzu fluorescent detector.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Preparation of the CS/DS Fractions from Shark Liver—GAGs were extracted from shark liver by protease digestion and alkali treatment, recovered by ethanol precipitation, and purified by anion exchange chromatography on a DEAE-Sephadex column. The GAG preparation thus obtained was treated with nitrous acid followed by gel filtration to remove heparin/HS (~50% of all GAG). No significant hyaluronic acid-derived oligosaccharides were found by anion exchange HPLC in the digests of the CS/DS preparations treated with CSase ABC or hyaluronidase SH (data not shown). This preparation was further fractionated by anion exchange chromatography using an AccellTM Plus QMA cartridge, which was eluted stepwise with buffers containing 0.2, 1.0, 1.5, and 2.0 M NaCl. Only trace amounts of CS/DS were detected in the fractions eluted with buffers containing 0.2 and 2.0 M NaCl. 80 and 20% of the total CS/DS were detected in the fractions eluted with buffers containing 1.0 and 1.5 M NaCl and designated as SL-CS/DS (1.0 M) and SL-CS/DS (1.5 M), respectively. These preparations were passed through a C18 cartridge to remove peptides.

Determination of the Molecular Mass of Shark Liver CS/DS—The molecular mass of SL-CS/DS preparations was determined by gel filtration (Fig. 1). Using the calibration curve generated with standard polysaccharides, the average molecular mass of SL-CS/DS (1.5 M) was estimated to be 75.7 kDa, whereas SL-CS/DS (1.0 M) gave a broader peak with a molecular mass ranging from 3.8 to 38.9 kDa. The distinct sizes of these two preparations may suggest different structures and functions. Interestingly, the mass of SL-CS/DS (1.5 M) was comparable with that of shark skin CS/DS (SS-CS/DS) preparations (28), and the large molecular mass may be characteristic of shark CS/DS.

Analysis of the Disaccharide Composition—The disaccharide composition of SL-CS/DS fractions was determined by digestion with CSases differing in specificity, followed by anion exchange HPLC. The two SL-CS/DS preparations were individually digested with CSase ABC, a mixture of CSases AC-I and AC-II, or CSase B. Each digest was labeled with a fluorophore, 2AB, for high sensitivity and resolution and analyzed by anion exchange HPLC. The analysis revealed a unique and heterogeneous disaccharide composition with diverse sulfation patterns for both SL-CS/DS (1.0 M) and SL-CS/DS (1.5 M) as shown in Fig. 2 and Table 1. Both fractions showed {Delta}4,5HexUA{alpha}1–3GalNAc ({Delta}O), {Delta}4,5HexUA{alpha}1–3GalNAc(6S) ({Delta}C), {Delta}4,5HexUA{alpha}1–3GalNAc(4S) ({Delta}A), {Delta}4,5HexUA(2S){alpha}1–3GalNAc(6S) ({Delta}D), {Delta}4,5HexUA(2S){alpha}1–3GalNAc(4S) ({Delta}B), and {Delta}4,5HexUA{alpha}1–3GalNAc(4S,6S) ({Delta}E) in varying proportions (Table 1), where 2S,4S, and 6S stand for 2-O-, 4-O-, and 6-O-sulfate and 4S,6S stands for 4,6-O-disulfate, respectively. Note that a small yet appreciable proportion of the {Delta}4,5HexUA(2S){alpha}1–3GalNAc-(4S,6S) ({Delta}T) unit was detected in SL-CS/DS (1.5 M). SL-CS/DS (1.0 M) was relatively low sulfated due to significant proportions of monosulfated disaccharides, {Delta}C (31.8%) and {Delta}A (33.1%), with a sulfate/disaccharide unit ratio of 1.18, whereas SL-CS/DS (1.5 M) was enriched with oversulfated disaccharides, {Delta}B (18%), {Delta}E (22.8%), and {Delta}T (1.8%), with a sulfate/disaccharide unit ratio of 1.43 (Table 1). Thus, an unique composition was revealed for SL-CS/DS (1.0 M) with three kinds of disulfated disaccharide units ({Delta}B, {Delta}D, and {Delta}E) and for SL-CS/DS (1.5 M) with small proportions of {Delta}D and {Delta}T in addition to significant proportions of {Delta}B and {Delta}E.


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  		TABLE 1
Disaccharide composition of the SL-CS/DS preparations

SL-CS/DS preparations purified from shark liver were digested with CSase ABC, CSase B, or a mixture of CSases AC-I and AC-II and individually analyzed by anion exchange HPLC after 2AB labeling as detailed under "Experimental Procedures."

 


Figure 1
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  		FIGURE 1.
Determination of the average molecular mass of SL-CS/DS preparations by gel filtration. Each SL-CS/DS preparation (5 µg as GlcUA) was loaded onto a Superdex 200 column calibrated with molecular mass markers as described under "Experimental Procedures" and eluted with 0.2 M NH4HCO3 at a flow rate of 0.3 ml/min. Fractions were monitored with 1,9-dimethylmethylene blue dye, and the average molecular mass was estimated using the calibration curve (inset). The void volume (Vo) and the total volume (Vt) were determined using dextrans (average mass, 170–200 kDa) and NaCl, respectively. The triangles and squares indicate the elution profiles of SL-CS/DS (1.0 M) and SL-CS/DS (1.5 M), respectively.

 
To discriminate GlcUA- or IdoUA-containing disaccharides in CS/DS chains, an analysis was also carried out using a mixture of CSases AC-I and AC-II, which specifically digest Gal-NAc-GlcUA linkages in the CS structure (42), and CSase B, which specifically attacks GalNAc-IdoUA linkages in the DS structure (43). Both digestions yielded most of the unsaturated disaccharide units except for {Delta}O, {Delta}C, and {Delta}T. Nonsulfated units appear to exist as O units rather than iO units. However, the fact that no {Delta}O was observed in the CSase B digests of the two preparations may be partially due to the resistant nature of the iO unit to CSase B (44) and may not necessarily indicate the absence of this unit. Interestingly, the rare B unit was demonstrated for both preparations, as in the case of SS-CS/DS (28), and may be a unique feature of shark CS/DS. Another interesting feature is the obvious presence of iC units in the 1.0 M fraction but not in the 1.5 M fraction, although no C units were found in either. It is interesting that the {Delta}T unit for SL-CS/DS (1.5 M) was almost completely recovered in the CSase B digest and not in the digest obtained with a mixture of CSases AC-I and AC-II, suggesting that {Delta}T was derived exclusively from IdoUA(2S){alpha}1–3GalNAc(4S,6S) (iT unit), not from GlcUA(2S)beta1–3GalNAc(4S,6S). Thus, the composition of both preparations, although highly heterogenous, is distinct.


Figure 2
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  		FIGURE 2.
Anion exchange HPLC of CSase digests of SL-CS/DS (1.5 M). SL-CS/DS (1.5 M) was digested with CSase ABC (A), a mixture of CSases AC-I and AC-II (B), or CSase B (C), and after 2AB labeling the digests were analyzed by HPLC on an amine-bound silica PA-03 column using a NaH2PO4 gradient (indicated by the dashed line). The elution positions of authentic 2AB-labeled unsaturated disaccharides are indicated by arrows. 1, {Delta}4,5HexUA{alpha}1–3GalNAc; 2, {Delta}4,5HexUA{alpha}1–3GalNAc(6S); 3, {Delta}4,5HexUA{alpha}1–3GalNAc(4S); 4, {Delta}4,5HexUA(2S){alpha}1–3GalNAc(6S); 5, {Delta}4,5HexUA(2S){alpha}1–3GalNAc(4S); 6, {Delta}4,5HexUA{alpha}1–3GalNAc(4S,6S); 7, {Delta}4,5HexUA(2S){alpha} 1–3GalNAc(4S,6S).

 
These results also clearly revealed a significantly higher proportion of GlcUA than IdoUA in SL-CS/DS (1.0 M) with a molar ratio of 1.56:1 (GlcUA/IdoUA), whereas SL-CS/DS (1.5 M) showed a higher proportion of IdoUA with a molar ratio of 3.35:1 (IdoUA/GlcUA). In this context, these two SL-CS/DS fractions have a unique GlcUA/IdoUA composition and might have distinct functional properties. In view of the importance of A/iA, B/iB, and E/iE units for the growth factor-binding and NOP activities of E-CS/DS-H (32), SL-CS/DS preparations rich in iA, iB, and iE/E units may exert strong biological activities in growth factor-binding and neuritogenesis.


Figure 3
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  		FIGURE 3.
Inhibitory effects of SL-CS/DS and commercial CS and DS preparations on the binding of PTN to the immobilized embryonic pig brain CS/DS fraction with high PTN binding activity. A fixed concentration of PTN (100 ng) was mixed with SL-CS/DS (1.0 M), SL-CS/DS (1.5 M), CS-A, CS-B, CS-C, CS-D, or CS-E as an inhibitor at a final concentration of 0.5 µg/ml and incubated for 15 min. The interaction of PTN with the immobilized embryonic pig brain CS/DS fraction, which has high affinity for PTN (E-CS/DS-H), was analyzed using a BIAcore J system as described under "Experimental Procedures." Results are expressed as relative percentages of inhibition compared with the binding of PTN to E-CS/DS-H in the absence of inhibitors.

 
Demonstration of Strong Binding of PTN to SL-CS/DS—To investigate biological activities of the purified SL-CS/DS preparations, their possible inhibitory activity was evaluated against the binding of PTN to the PTN-high affinity fraction of embryonic pig brain-derived CS/DS (E-CS/DS-H) using a BIAcore system (Fig. 3). This combination of PTN and CS/DS was used to evaluate biological activities, because E-CS/DS-H interacts strongly with PTN and also promotes the outgrowth of neurites in hippocampal neurons in vitro (32). Compared with various commercial CS and DS preparations, such as CS-E (sulfate/disaccharide unit ratio = 1.53), SL-CS/DS (1.5 M) showed stronger inhibition of the PTN binding to E-CS/DS-H (Fig. 3). In strong contrast, SL-CS/DS (1.0 M) showed no significant inhibition. These results have suggested the presence of a PTN-binding domain in the SL-CS/DS (1.5 M) but not in the SL-CS/DS (1.0 M) chains.

Demonstration of the Binding Activity of SL-CS/DS (1.5 M) toward Various Growth Factors Expressed during Brain Development—Based on the finding that SL-CS/DS (1.5 M) strongly binds PTN, we speculated that this SL-CS/DS preparation might interact with other growth factors involved in the brain's development. Hence, an analysis was carried out using the BIAcore system. The purified SL-CS/DS (1.5 M) preparation was biotinylated and immobilized on the streptavidin-precoated sensor chip. To determine the association and dissociation rate constants (ka and kd) as well as the dissociation equilibrium constants (Kd), various heparin-binding growth factors (PTN, MK, HB-EGF, VEGF165, HGF, FGF-7, or FGF-1) were injected at different concentrations individually onto the surface of a sensor chip coated with SL-CS/DS (1.5 M). Overlaid sensorgrams are shown in Fig. 4. These sensorgrams were analyzed collectively by using "the 1:1 Langmuir binding model with mass transfer" of the BIAevaluation 3.1 software to calculate the kinetic parameters. The kinetic parameters are summarized in Table 2 for all the growth factors except FGF-1, which exhibited only a weak binding response.


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  		TABLE 2
Kinetic parameters for the interaction of growth factors with immobilized SL-CS/DS (1.5 M)

The ka, kd, and Kd values were determined using a 1:1 Languimuir binding model with mass transfer as described under "Experimental Procedures." The value for each growth factor is expressed as the mean ± S.E. of five different concentrations.

 
The tested growth factors varied in their ability to bind SL-CS/DS (1.5 M). HGF, FGF-2, MK, and PTN displayed quick binding and a slow dissociation, giving Kd values in the low nanomolar range and signifying their strong affinity for SL-CS/DS (1.5 M) (Table 2). In contrast, HB-EGF, VEGF165, and FGF-7 showed weaker affinity for SL-CS/DS (1.5) as reflected in the Kd values listed in Table 2. These differences in affinity support the specificity of the interactions between SL-CS/DS (1.5 M) chains and various heparin-binding growth factors, suggesting a biological significance of these interactions.

NOP Activity of the SL-CS/DS Preparation—That SL-CS/DS (1.5 M) chains specifically interacted with some of the growth factors involved in the brain's development suggests that they may possess NOP activity. To evaluate the NOP activity of SL-CS/DS preparations, embryonic day 16 mouse hippocampal neuronal cells were utilized. The cells were cultured on a substrate coated with SL-CS/DS (1.5 M), SL-CS/DS (1.0 M), CS-E (a positive control), or P-ORN alone (a negative control). The length of the longest neurite of each of 100 randomly selected cells cultured on each substrate was measured. The neuronal cells cultured on the P-ORN substrate had some short neurites; however, their length was not significant (Fig. 5, A and bottom). In contrast, the neuronal cells cultured on coverslips precoated with SL-CS/DS (1.5 M) exhibited striking NOP activity (Fig. 5, D and bottom), showing neurites axonic in nature with stronger activity than a positive control CS-E (Fig. 5, B and bottom). It is interesting that although SL-CS/DS (1.5 M) and CS-E have comparable sulfate/unit ratios, 1.43 and 1.53, respectively, the former displayed stronger activity, suggesting that the types and sequential arrangement of oversulfated disaccharides are important. In contrast, SL-CS/DS (1.0 M) (Fig. 5, C and bottom) showed weak yet significant NOP activity stronger than the negative control of P-ORN (Fig. 5, A and bottom). These results suggest that the two preparations from SL-CS/DS exert stimulatory effects on hippocampal neurons to different extents.


Figure 4
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  		FIGURE 4.
Binding of various growth factors to immobilized SL-CS/DS (1.5 M). Various concentrations of PTN (A), MK (B), HB-EGF (C), VEGF165 (D), HGF (E), FGF-7 (F), FGF-2 (G), or FGF-1 (H) were injected onto the surface of an SL-CS/DS (1.5 M)-immobilized sensor chip, and the sensorgrams obtained with various concentrations of each growth factor were overlaid using BIAevaluation software (version 3.1). RU, resonance units.

 


Figure 5
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  		FIGURE 5.
Neurite outgrowth-promoting activities of SL-CS/DS preparations. E16 mouse hippocampal neuronal cells (10,000 cells/cm2) were grown for 24 h on various substrates precoated with P-ORN (A) and subsequently with CS-E (B) as a positive control, SL-CS/DS (1.0 M)(C), or SL-CS/DS (1.5 M)(D), fixed, and immunostained for microtubule-associated protein 2 and neurofilament. Bottom, 100 randomly selected individual neurons were used to measure the mean length of the longest neurite under each set of conditions. The values obtained from two separate experiments are expressed as the mean ± S.E. Mann-Whitney's U test was used to evaluate the significance of differences between the means (**, p < 0.01; ***, p < 0.001).

 
Investigation of the Distribution of GlcUA and IdoUA in the SL-CS/DS (1.5 M) Chains—Previously, our group showed IdoUA-containing CS/DS hybrid structures for E-CS/DS (26), hagfish notochord CS-H (27), and SS-CS/DS (28) as a key functional structure for the growth factor-binding and NOP activities. Therefore, to investigate the structural basis of the growth factor-binding and NOP activities of SL-CS/DS (1.5 M), the preparation was digested with various CSases differing in specificity, and a comparison of the resultant fragments was made by gel filtration. CSase ABC catalyzes the eliminative cleavage of most if not all the galactosaminidic linkages in CS/DS chains to produce disaccharides, resulting in a nearly complete digestion of SL-CS/DS (1.5 M) (Fig. 6A). In contrast, a mixture of CSases AC-I and AC-II, which specifically cleaves N-acetylgalactosaminidic linkages to give GlcUA, released 13.2% of all disaccharides compared with CSase ABC (Table 1; note the difference in the scale of Fig. 6A), and the majority of the resistant fragments ranged greatly in size from tetrasaccharides to polysaccharides (Fig. 6B). In contrast, SL-CS/DS (1.5 M) was highly sensitive to CSase B, which cleaves the linkage between N-acetylgalactosamine and IdoUA, and the digest mainly gave disaccharides and tetrasaccharides with a small hexasaccharide peak, suggesting a higher proportion of IdoUA than GlcUA (Fig. 6C). These results clearly suggest the hybrid nature of SL-CS/DS (1.5 M) with shorter CS domains distributed among the AC-I-resistant and AC-II-resistant DS domains of various lengths.


Figure 6
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  		FIGURE 6.
Analysis of the distribution of CS and DS domains in SL-CS/DS (1.5 M) chains on a Superdex peptide column. The purified SL-CS/DS (1.5 M) (1 µg as GAG) was digested with CSase ABC (A), a mixture of CSases AC-I and AC-II (B), or CSase B (C), and digests were individually labeled with 2AB and analyzed on a Superdex peptide column as described under "Experimental Procedures." Vo, void volume. The total volume (Vt) was 24 ml (determined as described in the legend to Fig. 1). The elution positions of 2AB-labeled authentic unsaturated CS-derived standard disaccharides/oligosaccharides determined earlier (22) are indicated by arrows as follows: 1, CS-decasaccharides; 2, CS-octasaccharides; 3, CS-hexasaccharides; 4, CS-tetrasaccharides; 5, trisulfated CS-disaccharides; 6, disulfated CS-disaccharides; 7, monosulfated CS-disaccharides; 8, nonsulfated CS-disaccharides.

 
Contribution of the DS-like Structure to the Growth Factor-binding and NOP Activities of SL-CS/DS (1.5 M)—The sequential arrangement of CS and DS domains appears to be critical to the growth factor-binding and NOP activities of SL-CS/DS (1.5 M). To clarify this viewpoint, the bioactivities of digests of SL-CS/DS (1.5 M) obtained with CSase ABC, a mixture of CSases AC-I and AC-II, or CSase B were further evaluated, first by assaying inhibition of the binding of growth factors to the SL-CS/DS-immobilized sensor chip using a BIAcore system. As shown in Fig. 7, top, digestion of SL-CS/DS (1.5 M) with CSase ABC or B almost completely abolished its inhibitory activity against most of the growth factors binding to SL-CS/DS. In contrast, only 10–30% inhibition was observed with a CSase AC-I/AC-II digest for all of the ligands used. Subsequently, NOP assays were carried out to confirm the different contributions of the CS and DS moieties in SL-CS/DS (1.5 M). Enzyme digests were individually coated on P-ORN-precoated coverslips, and then hippocampal neuronal cells were cultured (Fig. 7, bottom). As expected, the NOP activity of SL-CS/DS (1.5 M) was completely eliminated by digestion with CSase ABC or B, and the activity was dramatically reduced to the basal level, comparable with that for P-ORN alone, whereas digestion with a mixture of CSases AC-I and AC-II resulted in only a partial loss of the activity (Fig. 7, bottom). The results confirmed the key role of IdoUA-containing DS domains in the growth factor-binding and NOP activities of SL-CS/DS (1.5 M).

Investigation of the Involvement of 2-O-Sulfate in the NOP Activity of SL-CS/DS (1.5 M)—The sulfation pattern of CS/DS is crucial to the CS/DS-mediated bioactivities. To investigate the roles of 2-O-sulfation in the NOP activity of SL-CS/DS (1.5 M) in view of the high proportion (18%) of the iB unit, SL-CS/DS (1.5 M) was desulfated by the alkali treatment (see "Experimental Procedures"), and the resultant preparation was used for coating P-ORN-precoated coverslips for the NOP assay (Fig. 8). The alkali treatment, which removes 2-O-sulfate from the IdoUA of heparin (39), was used to remove 2-O-sulfate groups from the SL-CS/DS (1.5 M) fraction as described under "Experimental Procedures." The disaccharide analysis of the treated sample showed 88.3% desulfation at 2-O-sulfate of {Delta}B and a corresponding increase in the {Delta}A unit, with a concomitant decrease (45.8%) in {Delta}E resulting in the corresponding increase in {Delta}C (Table 3). Considering that monosulfated disaccharide units are resistant to the alkaline treatment, the changes in proportion of {Delta}4,5HexUA{alpha}1–3GalNAc(6S) and {Delta}4,5HexUA{alpha}1–3GalNAc (4S) seem to be primarily due to the desulfation of disulfated units (Table 3). The NOP of SL-CS/DS (1.5 M) toward hippocampal neurons was significantly suppressed by desulfation with the alkali treatment (Fig. 8) as compared with that of the untreated SL-CS/DS (1.5 M). These results suggested that oversulfated disaccharides, especially iB as well as iE/E units, are key elements for the NOP activity of SL-CS/DS (1.5 M), with the iB unit seeming to play a more important role than iE/E. Taken together, the DS and/or DS/CS domains containing oversulfated disaccharides, iB and/or iE/E, might be involved in forming functional sequences for the NOP activity of SL-CS/DS (1.5 M).


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  		TABLE 3
Comparison of disaccharide composition and degree of sulfation of SL-CS/DS (1.5 M) before and after alkali treatment

SL-CS/DS (1.5 M) (20 µg as GAGs) was treated with 0.1 M NaOH, and one-tenth of the resultant product and 0.5 µg of native preparation were individually digested with CSase ABC. Each digest was labeled with 2AB and analyzed by anion exchange HPLC as described under "Experimental Procedures." ND, not determined.

 


Figure 7
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  		FIGURE 7.
Effects of digestion with CSases on the growth factor-binding and NOP activities of SL-CS/DS (1.5 M). Top, inhibition experiments were performed on the binding of various growth factors to immobilized SL-CS/DS (1.5 M) using CSase digests of SL-CS/DS (1.5 M) as inhibitors essentially as described in the legend to Fig. 2. SL-CS/DS (1.5 M) was digested with CSase ABC, CSase B, or a mixture of CSases AC-I and AC-II. Each digest (70 ng) was incubated with various growth factors (100 or 50 ng) in 70 µl of the running buffer for 15 min, and then the mixture was injected onto the surface of the SL-CS/DS (1.5 M)-immobilized sensor chip. Results are expressed as relative percentages of inhibition based on the inhibition of the binding of each growth factor to the SL-CS/DS (1.5 M)-immobilized sensor chip compared with the binding obtained in the absence of inhibitors. Bottom, CSase digests of SL-CS/DS (1.5 M) were used for the neurite outgrowth promotion analysis as described in the legend to Fig. 5.

 
Demonstration of the HGF and PTN Signaling Pathways in the NOP Activity of SL-CS/DS (1.5 M)—SL-CS/DS (1.5 M) specifically interacts with HGF, MK, PTN, and bFGF, which have neurotrophic activity and are widely expressed as autocrine and paracrine pleiotropic factors in the distinct neuronal cell populations of the developing and adult brain, including hippocampal neuronal cells (45–49). Hence, these endogenous heparin-binding growth factors may be involved in the mechanism of the expression of the NOP activity of SL-CS/DS (1.5 M). To examine this possibility, antibodies against HGF, PTN, MK, or bFGF were individually added to the system for the neutralization assay of the NOP activity of SL-CS/DS (1.5 M) toward hippocampal neurons from an embryonic mouse brain. The addition of anti-HGF or anti-PTN antibody markedly suppressed the NOP activity of SL-CS/DS (1.5 M) to the basal level of P-ORN (Fig. 9A). In strong contrast, the anti-MK or bFGF antibody showed no significant inhibition. These results suggest that the NOP activity of SL-CS/DS (1.5 M) is selectively mediated by HGF and PTN, whereas MK and bFGF do not mediate the NOP activity of SL-CS/DS and may not be directly involved in the neuritogenesis of hippocampal neuronal cells through interactions with brain CS/DS chains either.


Figure 8
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  		FIGURE 8.
Effect of desulfation on the NOP activity of SL-CS/DS (1.5 M). SL-CS/DS (1.5 M) was treated with 0.1 M NaOH for desulfation as described under "Experimental Procedures." The native and treated samples were assayed for NOP activity as described in the legend to Fig. 5. **, p < 0.01; ***, p < 0.001 (for the significance of differences, see the legend to Fig. 5).

 


Figure 9
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  		FIGURE 9.
Effects of antibodies against growth factors and their receptors on the NOP activity of SL-CS/DS (1.5 M) and E-CS/DS. E16 mouse hippocampal neuronal cells were seeded on P-ORN-coated coverslips precoated with SL-CS/DS (1.5 M)(A) or E-CS/DS (B). 2 h after the seeding, anti-HGF (3 µg/ml), anti-PTN (10 µg/ml), anti-MK (20 µg/ml), or anti-bFGF (10 µg/ml) was added to the culture, and the mean length of the longest neurite was evaluated under each set of conditions as described in the legend to Fig. 5. Since antibodies against HGF and PTN inhibited the NOP activity of the SL-CS/DS (1.5 M) or E-CS/DS preparation, anti-HGF receptor and anti-PTN receptor (ALK) antibodies were also tested separately. In parallel, the same amount of a IgG fraction purified from the corresponding host animal (mouse, goat, or rabbit) was used as a control, which showed no significant inhibition in the mean length of the longest neurite measured for SL-CS/DS (1.5 M) or E-CS/DS without the antibodies (data not shown). **, p < 0.01; ***, p < 0.001 (For the significance of differences, see the legend to Fig. 5).

 
To prove the involvement of the signaling pathways of HGF and PTN in the NOP activity of the CS/DS chains, effects of the antibodies against the receptors for HGF (c-Met) and for PTN (ALK) were investigated. The anti-c-Met and anti-ALK antibodies inhibited the NOP activity of SL-CS/DS (1.5 M) by 60 and 56%, respectively, revealing the involvement of the HGF and PTN signaling pathways in the NOP activity of the sugar chains (Fig. 9A).

To investigate the in vivo mechanism of the NOP activity of brain CS/DS, SL-CS/DS (1.5 M) was replaced by E-CS/DS, which was prepared from embryonic pig brain (26), in the above mentioned inhibition assay using the antibodies against the growth factors and receptors. The NOP activity of E-CS/DS was also strongly suppressed by the antibody against HGF, PTN, c-Met, and ALK by 58, 45, 47, and 38%, respectively, but barely by the antibody against MK or bFGF (Fig. 9B). These results suggest the involvement of the signaling pathways of HGF and PTN in the expression of the NOP activity of the embryonic brain CS/DS in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The growth factor-binding activities of CS/DS chains exhibit a positive correlation with their NOP activity. Some oversulfated CS and DS, which can interact with various brain-derived growth factors and neurotrophic factors, show significant NOP activities (24, 25, 27, 28). In this study, it was demonstrated for the first time that both the signaling pathways of PTN-ALK and HGF-cMet are involved in the NOP activities of CS/DS hybrid chains isolated from E16 embryonic mouse brain and of SL-CS/DS (1.5 M). Pleiotrophin uses protein-tyrosine phosphatase {zeta} and ALK as its receptors. Although the former, which is a CS proteoglycan, has been shown to be involved in neuritogenesis through the CS chains (31), ALK has also been demonstrated here to be involved in the NOP activity of E-CS/DS. Notably, E-CS/DS and SL-CS/DS chains immobilized on the P-ORN-coated substrate served as a scaffold to recruit endogenous PTN and HGF in the culture system and to stimulate neuronal cells, promoting neuritogenesis.

PTN, MK, HGF, and bFGF, examined in this study, are broadly expressed in the brain, including hippocampal neuronal progenitor cells, and implicated in the development of the brain (45–49). The NOP activity of SL-CS/DS (1.5 M) and E-CS/DS was significantly suppressed by antibodies against PTN or HGF and also by antibodies against their respective receptors (ALK and cMet) but not by anti-MK or anti-bFGF antibody. These results suggested that endogenous PTN and HGF were recruited by E-CS/DS and SL-CS/DS and mediated their NOP activities. Although ALK has been identified as a common receptor for PTN (50) and MK (51), it appears to be involved in the NOP activity of CS/DS through PTN signaling. MK and bFGF may be important for the survival or adhesion of neuronal cells. Anti-HGF antibody showed much stronger inhibitory activity than anti-PTN antibody, suggesting that HGF plays a more crucial role in the CS/DS-generated signaling to promote neuritogenesis.

HGF is a pleiotropic factor. It binds to and activates the tyrosine kinase receptor cMet and is also an axonal chemoattractant and neurotrophic factor for motor neurons (52, 53). It is required for the axonal growth of dorsal root ganglia sensory neurons (54) and elicits multiple functions in sympathetic neurons (55–57). HGF signaling potentiates the response of different neurons to specific signals (52). GAGs are essential co-receptors for the activation of cMet (58), since HGF can bind HS (59) or DS (60) chains of proteoglycans and also interacts with cMet to form an active ternary complex (61). In this study, CS/DS hybrid chains were demonstrated to recruit a minute amount of endogenous HGF to stimulate the outgrowth of neurites in hippocampal neurons, which may suggest that CS/DS presented HGF to cMet or both cMet and glycan co-receptors (HS or CS/DS) on the neuronal surface. The structure of the HGF-binding sites on HS and CS/DS chains remains to be investigated.

PTN is expressed at the intracellular matrix of axonal tracts in the developing brain (30, 62, 63) and involved in the development of axons in vivo and the outgrowth of neurites in vitro (29, 64). PTN induces neurite outgrowth through specific interaction with the HS side chains of syndecan-3 (65), although our studies have shown that PTN also interacts with both endogenous and exogenous CS/DS chains (21, 26, 27, 38). The oversulfated disaccharide units in CS/DS chains are critical to such interaction. However, it remains unclear which oversulfated disaccharide plays a major role in the binding of PTN. An analysis of the PTN-binding fractions of E-CS/DS (32) or SS-CS/DS4 showed a significant increase in the proportion of {Delta}B and {Delta}E with increased affinity for PTN, whereas the proportion of {Delta}D decreased. Similarly, SL-CS/DS (1.5 M), which showed high proportions of {Delta}B and {Delta}E units, strongly inhibited the binding of PTN to E-CS/DS-H, whereas CS-D displayed weak inhibition. Hence, the B/iB and E/iE units in CS/DS chains, which are the parental structures for {Delta}B and {Delta}E, are more important to the binding of PTN than D/iD units.

The drastic abolishment of the growth factor-binding and NOP activities (Fig. 7) of SL-CS/DS (1.5 M) by digestion with CSase B rather than a mixture of CSases AC-I and AC-II revealed the key role of IdoUA-containing disaccharides. The critical role of IdoUA has also been demonstrated for the growth factor-binding and NOP activities of relatively low sulfated CS/DS preparations, including E-CS/DS (sulfate/disaccharide unit ratio = 0.83) (26) and SS-CS/DS (sulfate/disaccharide unit ratio = 1.17) (28). These and other findings together support the notion that disulfated units and IdoUA are critical factors for these activities. Although the data strongly suggest their involvement, the final proof awaits a demonstration of IdoUA in the functional domains for the growth factor-binding and neurite outgrowth-promoting activities. Compared with GlcUA, IdoUA can form various conformations, resulting in an inherent plasticity for interaction with various protein partners (66, 67). However, the low sulfated porcine skin DS, or CS-B, which is rich in iA units (IdoUA{alpha}1–3GalNAc(4S)), is a weak inhibitor for the binding of PTN to E-CS/DS-H, which in turn indicates the requirement for IdoUA-containing oversulfated disaccharides, such as IdoUA(2S){alpha}1–3GalNAc(4S) (iB) and IdoUA{alpha}1–3GalNAc(4S,6S) (iE). In this context, adult sea urchin DS, which contains proportions of iB and iE comparable with those of SL-CS/DS (1.5 M), exhibited weaker NOP activity than SL-CS/DS (1.5 M) and CS-E, which contains 56% E units but no iB units (25). Interestingly, adult sea urchin DS contains 59% iC but only less than 1% iA, whereas SL-CS/DS (1.5 M) contains 42.6–49.4 mol % of iA. In addition, {Delta}A was observed with increasing proportions of {Delta}B and {Delta}E in the high PTN affinity fraction of E-CS/DS and SS-CS/DS4 and with a decrease in {Delta}C. The greater NOP activity of SL-CS/DS (1.5 M) than CS-E (Fig. 5) suggests that iB, iE, and iA units are preferred for the PTN binding activity and also for the NOP activity of CS/DS chains.

Previously, it was demonstrated that anti-PTN antibody strongly suppressed the endogenous PTN-mediated NOP activity of E-CS/DS chains with low affinity for PTN (E-CS/DS-L) but did not significantly inhibit that of E-CS/DS with high affinity for PTN (E-CS/DS-H) (32). Hence, it seems that PTN mediates the NOP activity of E-CS/DS-L but not E-CS/DS-H. A series of octasaccharides containing at least one D unit have been isolated from E-CS/DS-L (32). Although a signaling molecule responsible for the NOP activity of E-CS/DS-H remains to be identified, the possibility exists that PTN and other growth factors share the binding sites in E-CS/DS-H chains. The binding of other growth factors to the putative overlapping binding sites on E-CS/DS-H chains may be involved in the neuritogenesis. The specific interaction of SL-CS/DS (1.5 M) with PTN, MK, HGF, and bFGF implies the existence of such overlapping binding sites for these growth factors. HGF is a strong candidate for a factor involved in the NOP activity of E-CS/DS-H.

SL-CS/DS (1.5 M) is a potential candidate for a non-mammal-derived therapeutic agent. Further investigation of the functional domains of SL-CS/DS (1.5 M) involved in PTN and HGF signaling should provide a basis for developing specific oligosaccharide drugs with fewer side effects than the parental polysaccharides.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid 17659020, 16390026, and 14082207 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Core Research for Evolutional Science and Technology program of the Japan Science and Technology Agency, and the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by a postdoctoral fellowship from the Japan Society for the Promotion of Science. Back

2 To whom correspondence should be addressed: Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Nishi 11-choume, Kita 21-jo, Kita-ku, Sapporo, Hokkaido 001-0021, Japan. Tel.: 81-11-706-9054; Fax: 81-11-706-9056; E-mail: k-sugar{at}sci.hokudai.ac.jp.

3 The abbreviations used are: GAG, glycosaminoglycan; CS, chondroitin sulfate; DS, dermatan sulfate; SL-CS/DS, shark liver chondroitin sulfate/dermatan sulfate; E-CS/DS, embryonic pig brain-CS/DS; E-CS/DS-H, E-CS/DS high affinity fraction; SS-CS/DS, shark skin chondroitin sulfate/dermatan sulfate; IdoUA, L-iduronic acid; GlcUA, D-glucuronic acid; FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; rh, recombinant human; E16, embryonic day 16; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PTN, pleiotrophin; MK, midkine; VEGF165, vascular endothelial growth factor-165; HGF/SF, hepatocyte growth factor/scatter factor; HexUA, hexuronic acid; {Delta}4,5HexUA, 4,5-unsaturated hexuronic acid; 2AB, 2-aminobenzamide; P-ORN, poly-DL-ornithine; HPLC, high performance liquid chromatography; CSase, chondroitinase; NOP, neurite outgrowth-promoting. Back

4 F. Li, C. D. Nandini, X. Bao, T. Nakamura, T. Muramatsu, and K. Sugahara, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Toshikazu Nakamura (Osaka University) for the anti-rat HGF rabbit IgG and technical advice.



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 ABSTRACT
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 DISCUSSION
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