Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)
Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)
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DOI：
10.1093/glycob/10.3.273
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发表时间：
2000-03-01
期刊：
影响因子：
4.3
通讯作者：
Midura, RJ
中科院分区：
文献类型：
--
作者：
Calabro, A;Benavides, M;Midura, RJ
Hybryonan和软骨素/硫酸皮肤素是糖胺聚糖，在包括软骨在内的各种组织的生物力学特性中发挥主要作用。软骨素/硫酸皮肤素链可分为三个区域：（1）单个连接区域寡糖，通过该区域链连接到其蛋白聚糖核心蛋白，（2）许多内部重复二糖，其构成链的大部分，和（3）单个非还原性末端糖结构。软骨素/硫酸皮肤素链的这些区域中的每一个都具有其自身的结构微异质性水平，其随蛋白聚糖类、组织来源、物种和病理而变化。我们已经开发出快速，简单，灵敏的协议，检测，表征和定量的糖结构的内部二糖和非还原性末端区域的透明质酸和软骨素/硫酸皮肤素链。这些方案依赖于通过特定的酶处理（透明质酸酶/软骨素酶）产生具有游离还原基团的糖结构，然后通过其游离还原基团用荧光报告基因2-氨基吖啶酮进行定量标记。这些糖结构的进一步特征在于通过额外的酶（硫酸酯酶）或化学（汞离子）处理进行修饰。在通过荧光团辅助的碳水化合物电泳分离后，用冷却的电荷耦合器件照相机定量每个条带中的相对荧光以进行分析。具体地，鉴定的消化产物是（1）不饱和内部δ二糖，包括δ DiHA、δ Di 0 S、δ Di 2S、δ Di 4S、δ Di 6S、δ Di 2，4S、δ Di 2，6S、δ Di 4，6S和δ Di 2，4，6S;（2）饱和非还原末端二糖，包括DiHA、DIGS、Di 4S和Di 6S;和（3）非还原性末端己糖胺，包括glcNAc、galNAc、4S-galNAc、6S-galNAc和4，6S-galNAc。
Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Delta disaccharides including Delta DiHA, Delta Di0S, Delta Di2S, Delta Di4S, Delta Di6S, Delta Di2,4S, Delta Di2,6S, Delta Di4,6S, and Delta Di2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, DIGS, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4,6S-galNAc.