Karamanos N K, Aletras A J, Tsegenidis T, Tsiganos C P, Antonopoulos C A
Department of Chemistry, University of Patras, Greece.
Eur J Biochem. 1992 Mar 1;204(2):553-60. doi: 10.1111/j.1432-1033.1992.tb16667.x.
Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.
通过离子交换色谱、凝胶色谱和密度梯度离心从4M盐酸胍提取物中分离出鱿鱼皮中的过硫酸化硫酸软骨素蛋白聚糖。该蛋白聚糖的分子量为3.5×10⁵，平均含有六条过硫酸化硫酸软骨素链（分子量为4×10⁴），这些链结合在一条分子量为2.8×10⁴的多肽上，并且含有由己糖胺、葡萄糖醛酸、硫酸盐和岩藻糖（作为唯一的中性单糖）组成的寡糖。该蛋白聚糖蛋白核心的主要氨基酸是甘氨酸（约占总氨基酸的三分之一）、天冬氨酸/天冬酰胺和丝氨酸，它们总共占总量的50%。该蛋白聚糖对蛋白水解酶V8蛋白酶、胰蛋白酶（用二苯基甲酰氯处理）、α-胰凝乳蛋白酶和链霉蛋白酶具有抗性，而它会被木瓜蛋白酶完全降解，并在很大程度上被胶原酶降解。用软骨素酶AC预处理的蛋白聚糖会被链霉蛋白酶在很大程度上降解，并被V8蛋白酶和胰蛋白酶轻微降解。该蛋白聚糖不与透明质酸相互作用，也不形成自聚集体。过硫酸化硫酸软骨素链由不寻常的硫酸化二糖单元组成，这些单元通过高效液相色谱法进行分离和表征。特别地，它含有2-乙酰氨基-2-脱氧-3-O-(α-L-苏式-4-烯吡喃糖醛酸)-D-半乳糖4-硫酸盐（δ二糖-4S）和二硫酸化二糖（δ二糖-二S）[90%的2-乙酰氨基-2-脱氧-3-O-(α-L-苏式-4-烯吡喃糖醛酸2/3-硫酸盐)-D-半乳糖6-硫酸盐（δ二糖-二SD）和10%的2-乙酰氨基-2-脱氧-3-O-(α-L-苏式-4-烯吡喃糖醛酸2/3-硫酸盐)-D-半乳糖4-硫酸盐（δ二糖-二SK）]作为主要二糖，大量的三硫酸化二糖（δ二糖-三S）以及少量的2-乙酰氨基-2-脱氧-3-O-(α-L-苏式-4-烯吡喃糖醛酸)-D-半乳糖6-硫酸盐（δ二糖-6S）和2-乙酰氨基-2-脱氧-3-O-(α-L-苏式-4-烯吡喃糖醛酸)-D-半乳糖（δ二糖-0S）。三硫酸化二糖在半乳糖胺的C-4和C-6以及葡萄糖醛酸的C-2或C-3处含有硫酸基团。通过对纯的过硫酸化硫酸软骨素制剂进行高效液相色谱分析，发现它最有可能含有葡萄糖、半乳糖、甘露糖和岩藻糖作为分支。
