Berry M, Ellingham R B, Corfield A P
Department of Ophthalmology, University of Bristol, Bristol Eye Hospital, United Kingdom.
Invest Ophthalmol Vis Sci. 1996 Dec;37(13):2559-71.
To isolate all constituent mucins from human conjunctival mucus.
Mucins were extracted from human conjunctiva in guanidine hydrochloride and protease inhibitors. The mucins were isolated by density gradient centrifugation, gel filtration, and ion exchange chromatography. Throughout purification, the mucin profile was monitored by agarose electrophoresis and vacuum blotting. Blots were probed for peptide and carbohydrate epitopes. The latter included IE3 and TKH2 specific for Tn and sialyl-Tn, respectively, considered tumor-related antigens. In vivo impression cytology specimens of normal conjunctival goblet cells also were probed with the same reagents. Oligosaccharides were released from isolated mucins by alkaline beta-elimination and then size fractionated.
Human conjunctival mucins consist of at least three size populations; the largest is excluded on Sepharose CL2B. The two largest populations are polydisperse. Their overall electrophoretic pattern is conserved between individuals. Similar charge distributions were detected in different buoyant density ranges from the density gradient centrifugation: a less charged population containing three components and a highly charged population with two components on agarose electrophoresis. Cross-reaction with IE3 and TKH2 was detected throughout purification in the largest mucins, which were presumably mature, and in impression cytology. Oligosaccharides from mucins in each buoyant density were largely in the monosaccharide and disaccharide range, consistent with Tn and sialyl-Tn standards.
Secreted human conjunctival mucins are polydisperse, with discrete components appearing consistently in pooled and individual samples. They have a unique oligosaccharide pattern containing Tn and sialyl-Tn. This indicates normal roles in normal human ocular mucins for these antigens, which are disease markers in other tissues.
从人结膜黏液中分离出所有组成性黏蛋白。
在含有盐酸胍和蛋白酶抑制剂的条件下从人结膜中提取黏蛋白。通过密度梯度离心、凝胶过滤和离子交换色谱法分离黏蛋白。在整个纯化过程中，通过琼脂糖电泳和真空印迹法监测黏蛋白谱。对印迹进行肽和碳水化合物表位检测。后者包括分别对Tn和唾液酸化Tn特异的IE3和TKH2，它们被认为是肿瘤相关抗原。还用相同试剂检测了正常结膜杯状细胞的体内印迹细胞学标本。通过碱性β-消除法从分离出的黏蛋白中释放寡糖，然后进行大小分级分离。
人结膜黏蛋白至少由三种大小的群体组成；最大的群体被Sepharose CL2B排除在外。两个最大的群体是多分散的。它们的整体电泳模式在个体之间是保守的。在密度梯度离心的不同浮力密度范围内检测到相似的电荷分布：在琼脂糖电泳上，一个电荷较少的群体包含三个组分，一个电荷较多的群体包含两个组分。在整个纯化过程中，在可能成熟的最大黏蛋白以及印迹细胞学中均检测到与IE3和TKH2的交叉反应。每个浮力密度下黏蛋白的寡糖大多在单糖和二糖范围内，与Tn和唾液酸化Tn标准一致。
分泌的人结膜黏蛋白是多分散的，在合并样本和个体样本中始终出现离散的组分。它们具有包含Tn和唾液酸化Tn的独特寡糖模式。这表明这些在其他组织中作为疾病标志物的抗原在正常人眼黏蛋白中具有正常作用。
