Deepa Sarama Sathyaseelan, Kalayanamitra Kittiwan, Ito Yumi, Kongtawelert Prachya, Fukui Shigeyuki, Yamada Shuhei, Mikami Tadahisa, Sugahara Kazuyuki
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Biochemistry. 2007 Mar 6;46(9):2453-65. doi: 10.1021/bi602374m. Epub 2007 Feb 7.
A mixture of octa- and decasaccharides obtained by the digestion with the hyaluronidase of chondroitin sulfate E derived from squid cartilage was subfractionated into 20 and 23 different components, respectively, by anion-exchange HPLC. MALDI-TOF/MS was used to assign the sugar and sulfate composition of the putative octa- and decasaccharides, and a disaccharide composition analysis revealed the building blocks to be A- [GlcUAbeta1-3GalNAc(4S)], C- [GlcUAbeta1-3GalNAc(6S)], and E- [GlcUAbeta1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences of these octa- and decasaccharides were determined at low picomole amounts by a combination of enzymatic digestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that each fraction is a mixture of a major component together with one to three minor components, reflecting the heterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reported here, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of the sequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibody MO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-E decasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, only E-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end and also the importance of chain length, which in turn may indicate the importance of the conformation acquired by this specific sequence for antibody recognition.
用来自鱿鱼软骨的硫酸软骨素E经透明质酸酶消化得到的八糖和十糖混合物，通过阴离子交换高效液相色谱法分别亚分馏为20种和23种不同组分。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）用于确定假定的八糖和十糖的糖和硫酸盐组成，二糖组成分析表明其结构单元为A- [β-D-葡萄糖醛酸-1,3-N-乙酰半乳糖胺（4-O-硫酸酯）]、C- [β-D-葡萄糖醛酸-1,3-N-乙酰半乳糖胺（6-O-硫酸酯）]和E- [β-D-葡萄糖醛酸-1,3-N-乙酰半乳糖胺（4-O,6-O-二硫酸酯）]单元，其中4S和6S分别代表4-O-硫酸酯和6-O-硫酸酯。通过硫酸软骨素酶酶切结合阴离子交换高效液相色谱法，在低皮摩尔量下确定了这些八糖和十糖序列。测序表明，每个馏分都是一种主要成分与一到三种次要成分的混合物，反映了母体多糖的异质性。本文报道的11种不同八糖序列中，有8种是新的，而6种十糖序列全部是新的，这是关于比八糖更长的硫酸软骨素寡糖测序的首次报道。用寡糖微阵列检测单克隆抗体MO-225与八糖和十糖的反应性，结果表明硫酸软骨素E十糖是抗体识别的最小要求。在6种十糖中，只有E-E-E-E-C被MO-225识别，这表明还原端需要一个C单元，也表明了链长的重要性，这反过来可能表明这种特定序列所获得的构象对抗体识别的重要性。
