Cheng F, Yoshida K, Heinegård D, Fransson L A
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Glycobiology. 1992 Dec;2(6):553-61. doi: 10.1093/glycob/2.6.553.
We have developed a new procedure for the sequence analysis of glycosaminoglycans, which is particularly suitable for the analysis of chains from heavily substituted proteoglycans. The procedure has been applied to various aggrecan-derived chondroitin sulphates. The glycans are released from the core protein by alkaline scission of the xylose-serine bond, subjected to reductive amination using p-aminobenzoic acid and finally radioiodinated at an acidic pH. Sequence analysis is performed by using various enzymic degradations, partial or complete, followed by high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify segments extending from the labelled reducing end to the point of cleavage. By using chondroitin C lyase to identify the location of 6-O-sulphated hexosamines, we find that chondroitin sulphate from tracheal cartilage has its 6-O-sulphated repeats concentrated to the extreme non-reducing terminal portion of the chain. In chondroitin sulphates derived from intervertebral discs (nucleus pulposus), the 6-O-sulphated repeats have a biphasic distribution; they occur mostly near the linkage region (i.e. the reducing end), but also in the non-reducing portion of the chain. Chondroitin sulphate from nasal cartilage, which is mostly 4-O-sulphated, displays considerable heterogeneity in the linkage region. Three or possibly more charge variants are observed.
我们已开发出一种用于糖胺聚糖序列分析的新方法，该方法特别适用于分析高度取代的蛋白聚糖中的链。此方法已应用于各种源自聚集蛋白聚糖的硫酸软骨素。聚糖通过木糖 - 丝氨酸键的碱性断裂从核心蛋白中释放出来，使用对氨基苯甲酸进行还原胺化，最后在酸性pH下进行放射性碘化。序列分析通过使用各种部分或完全的酶促降解来进行，随后进行高分辨率聚丙烯酰胺凝胶电泳、印迹和放射自显影，以鉴定从标记的还原端延伸至切割点的片段。通过使用软骨素C裂解酶来鉴定6 - O - 硫酸化己糖胺的位置，我们发现气管软骨中的硫酸软骨素其6 - O - 硫酸化重复序列集中在链的极端非还原末端部分。在源自椎间盘（髓核）的硫酸软骨素中，6 - O - 硫酸化重复序列具有双相分布；它们大多出现在连接区附近（即还原端），但也出现在链的非还原部分。来自鼻软骨的硫酸软骨素主要是4 - O - 硫酸化的，在连接区显示出相当大的异质性。观察到三种或可能更多的电荷变体。
