Nadanaka S, Clement A, Masayama K, Faissner A, Sugahara K
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, Japan.
J Biol Chem. 1998 Feb 6;273(6):3296-307. doi: 10.1074/jbc.273.6.3296.
A mouse brain chondroitin sulfate (CS) proteoglycan, DSD-1-PG, bears the DSD-1 epitope and has neurite outgrowth promoting properties. Shark cartilage CS-C inhibits the interactions between the DSD-1-specific monoclonal antibody 473HD and the CS chains of the DSD-1-PG, which is expressed on the mouse glial cells (Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., and Schachner, M. (1994) J. Cell Biol. 126, 783-799). On the other hand, several hexasaccharides isolated from commercial shark cartilage CS-D, which contains a higher proportion of characteristic D units (GlcUA(2-sulfate)beta1-3GalNAc(6-sulfate)) as compared with CS-C, has the A-D tetrasaccharide sequence composed of an A disaccharide unit (GlcUAbeta1-3GalNAc(4-sulfate)) and a D disaccharide unit (Nadanaka, S. and Sugahara, K. (1997) Glycobiology 7, 253-263). In this study, the biological activities and the structure of shark cartilage CS-D were investigated. CS-D inhibited the interactions between monoclonal antibody 473HD and DSD-1-PG and also promoted neurite outgrowth of embryonic day 18 hippocampal neurons. Eight octasaccharide fractions were isolated from CS-D after partial digestion with bacterial chondroitinase ABC by means of gel filtration chromatography and anion-exchange high performance liquid chromotography to investigate the frequency and the arrangement of the A-D tetrasaccharide unit in the polymer sequence. Structural analysis performed by a combination of enzymatic digestions with 500-MHz 1H NMR spectroscopy demonstrated that the isolated octasaccharides shared the common core structure DeltaHexAalpha1-3GalNAcbeta1-4(GlcUAbeta1-3GalNAc)3 with four, five, and six sulfate esters at various hydroxyl groups in different combinations. In the structure, DeltaHexA and GlcUA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively. No D-D tetrasaccharide sequence was found, and discrete D disaccharide units were demonstrated exclusively as A-D tetrasaccharide units in either an A-D-A or an A-D-C hexasaccharide sequence in the five octasaccharides that represented about 5.0% (w/w) of the starting polysaccharides (C denotes the disaccharide GlcUAbeta1-3GalNAc(6-sulfate)). It remains to be determined whether such characteristic hexasaccharide sequences present in shark cartilage CS-D serve as functional domain structures recognized by some protein ligands.
一种小鼠脑硫酸软骨素（CS）蛋白聚糖DSD-1-PG，带有DSD-1表位并具有促进神经突生长的特性。鲨鱼软骨CS-C可抑制DSD-1特异性单克隆抗体473HD与小鼠神经胶质细胞上表达的DSD-1-PG的CS链之间的相互作用（法斯纳，A.，克莱门特，A.，洛赫特，A.，施特赖特，A.，曼德尔，C.，和沙赫纳，M.（1994年）《细胞生物学杂志》126卷，783 - 799页）。另一方面，从商业鲨鱼软骨CS-D中分离出的几种六糖，与CS-C相比，其特征性D单元（GlcUA(2 - 硫酸酯)β1 - 3GalNAc(6 - 硫酸酯)）比例更高，具有由A二糖单元（GlcUAbeta1 - 3GalNAc(4 - 硫酸酯)）和D二糖单元组成的A - D四糖序列（稻田，S.和菅原，K.（1997年）《糖生物学》7卷，253 - 263页）。在本研究中，对鲨鱼软骨CS-D的生物学活性和结构进行了研究。CS-D抑制单克隆抗体473HD与DSD-1-PG之间的相互作用，还能促进胚胎第18天海马神经元的神经突生长。用细菌软骨素酶ABC部分消化CS-D后，通过凝胶过滤色谱法和阴离子交换高效液相色谱法分离出八个八糖级分，以研究聚合物序列中A - D四糖单元的频率和排列。通过结合500兆赫兹1H核磁共振光谱进行的酶消化进行的结构分析表明，分离出的八糖具有共同的核心结构DeltaHexAα1 - 3GalNAcβ1 - 4(GlcUAbeta1 - 3GalNAc)3，在不同的羟基上有四个、五个和六个硫酸酯，以不同组合形式存在。在该结构中，DeltaHexA和GlcUA分别代表4 - 脱氧-α - L - 苏式 - 己 - 4 - 烯吡喃糖醛酸和葡萄糖醛酸。未发现D - D四糖序列，并且在代表起始多糖约5.0%（w/w）的五个八糖中，离散的D二糖单元仅作为A - D - A或A - D - C六糖序列中的A - D四糖单元出现（C表示二糖GlcUAbeta1 - 3GalNAc(6 - 硫酸酯)）。鲨鱼软骨CS-D中存在的这种特征性六糖序列是否作为某些蛋白质配体识别的功能域结构仍有待确定。
