Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from Embryonic Pig Brain, Which Contain a Higher Proportion of L-Iduronic Acid than Those from Adult Pig Brain, Exhibit Neuritogenic and Growth Factor Binding Activities

Xingfeng Bao,Shuji Nishimura,Tadahisa Mikami,Shuhei Yamada,Nobuyuki Itoh,Kazuyuki Sugahara

doi:10.1074/jbc.m310877200

Abstract

We have shown that over-sulfated chondroitin sulfate/dermatan sulfate (CS/DS) chains from various marine organisms exhibit growth factor binding activities and neurite outgrowth-promoting activities in embryonic mouse hippocampal neurons in vitro. In this study we demonstrated that CS/DS hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain. In contrast, the CS/DS preparation from adult pig brain showed considerably less activity to bind these growth factors and no neuritogenic activity. Structural analysis indicated that the average size of the CS/DS chains was similar (40 kDa) between these two preparations, but the disaccharide compositions differed considerably, with a significant proportion of l-iduronic acid (IdoUA)-containing disaccharides (8 approximately 9%) in the CS/DS chains from embryos but not in those from adults (<1%). Interestingly, both neurite outgrowth-promoting activity and growth factor binding activities of the CS/DS chains from embryos were abolished by digestion not only with chondroitinase ABC but also with chondroitinase B, suggesting that the IdoUA-containing motifs are essential for these activities. These findings imply that the temporal expression of CS/DS hybrid structures containing both GlcUA and IdoUA and binding activities toward various growth factors play important roles in neurogenesis in the early stages of the development of the brain.

Highlights

Chondroitin sulfate proteoglycans (CS-PGs)1 are complex macromolecules consisting of a protein core and at least one
In this study we demonstrated that chondroitin sulfate/ dermatan sulfate (CS/dermatan sulfate (DS)) hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain
In the case of the GAG chains in the phosphate-buffered saline (PBS)-soluble fraction, the 1.0 M NaCl-eluted fraction, which contained more than 90% of the CS/DS chains of the embryonic or adult pig brain-derived sample, was used for further purification

Summary

EXPERIMENTAL PROCEDURES

Animals—Embryonic pigs (body weight: 315 g) and adult pig brains were purchased from a local pig-raising company. ddY mice were used for the preparation of E16 hippocampal neurons. To remove hyaluronic acid (HA) PBS-soluble and -insoluble fractions obtained from both embryonic and adult pig brains were subjected to digestion with Streptomyces hyaluronidase at 60 °C in 20 mM acetate buffer, pH 5.0 [30]. Analysis of Disaccharide Composition—An aliquot (4.0 ␮g) of Es-, Ei-, As-, or Ai-CS/DS was incubated with 40 mIU of chondroitinase ABC in a 50 mM Tris-HCl buffer, pH 8.0, containing 60 mM sodium acetate in a total volume of 40 ␮l at 37 °C for 4 h [33]. To investigate the structural characteristics of the functional epitopes in Es-CS/DS responsible for its binding to various growth factors, one aliquot (5 ␮g) of biotinylated Es-CS/DS was digested with 10 mIU of chondroitinase ABC and another with 4 mIU of chondroitinase B as described above, and the resultant digest was immobilized on the sensor chip surface. The kinetic parameters were evaluated with BIAevaluation software 3.1 (BIAcore AB, Uppsala, Sweden) using a 1:1 binding model with mass transfer

RESULTS

CSase ABC

DISCUSSION

Kd nM