Chapman S A, Ayad S, O'Donoghue E, Bonshek R E
Department of Pathological Sciences, University of Manchester, UK.
Eye (Lond). 1998;12 ( Pt 3a):440-8. doi: 10.1038/eye.1998.102.
To analyse high-molecular-weight matrix glycoproteins in trabecular meshwork, cornea and sclera using SDS/PAGE and immuno- and lectin blotting.
Extracts of normal trabecular meshwork (TM), cornea and sclera were analysed under reducing conditions on SDS/ PAGE. Western blots were stained for total protein, and major high-molecular-weight components were identified by immunoblotting with antibodies to fibronectin (FN) and type VI collagen. Lectin blotting with PSA, MPA and DSA identified some of the glycoprotein glycans.
FN antibody bound to the 240 kDa band in TM, cornea and sclera. Type VI collagen antibody bound more strongly to one band and less so to two other bands at approximately 200 kDA in normal TM and to a ladder of bands in cornea and sclera. PSA and DSA bound at 240, 200 and 140 kDa in TM, cornea and sclera. MPA bound at 240, 200 and 140 kDa in TM and at 240, 200 and approximately 120 kDA in cornea and sclera.
FN is a component of the band at 240 kDA in TM, cornea and sclera. Normal TM was found to contain relatively more of one of the isoforms of the alpha 3 (VI) chain whilst cornea and sclera contained all the alpha 3 (VI) isoforms. Complex N-linked bi/tri-antennary glycans were localised in FN and the alpha 1, alpha 2 and alpha 3 (VI) chains in TM, cornea and sclera. O-linked glycans (identified by MPA binding) were located in FN and alpha 3 (VI) chains of TM, cornea and sclera.
采用十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）以及免疫印迹和凝集素印迹法分析小梁网、角膜和巩膜中的高分子量基质糖蛋白。
在还原条件下，对正常小梁网（TM）、角膜和巩膜的提取物进行SDS/PAGE分析。蛋白质免疫印迹法用于检测总蛋白，通过用抗纤连蛋白（FN）和VI型胶原抗体进行免疫印迹鉴定主要的高分子量成分。用前列腺特异性抗原（PSA）、甘露糖结合蛋白A（MPA）和双花扁豆凝集素（DSA）进行凝集素印迹法鉴定部分糖蛋白聚糖。
FN抗体与TM、角膜和巩膜中的240 kDa条带结合。VI型胶原抗体在正常TM中与约200 kDa的一条条带结合更强，与另外两条条带结合较弱，在角膜和巩膜中则与一系列条带结合。PSA和DSA在TM、角膜和巩膜中与240 kDa、200 kDa和140 kDa的条带结合。MPA在TM中与240 kDa、200 kDa和140 kDa的条带结合，在角膜和巩膜中与240 kDa、200 kDa和约120 kDa的条带结合。
FN是TM、角膜和巩膜中240 kDa条带的组成成分。发现正常TM中α3（VI）链的一种异构体相对较多，而角膜和巩膜中含有所有的α3（VI）异构体。复杂的N-连接双/三触角聚糖定位于TM、角膜和巩膜中的FN以及α1、α2和α3（VI）链。O-连接聚糖（通过MPA结合鉴定）位于TM、角膜和巩膜的FN以及α3（VI）链中。
