Plantner J J
Lorand V. Johnson Laboratory for Research in Ophthalmology, Department of Surgery, Case Western Reserve University, Cleveland, OH 44106.
Exp Eye Res. 1992 Jan;54(1):113-25. doi: 10.1016/0014-4835(92)90075-4.
A very high molecular weight mucin-like glycoprotein was isolated by gel filtration of interphotoreceptor matrix (IPM) from fresh bovine eyes and purified to apparent homogeneity by cesium chloride/guanidine hydrochloride (GuHCl) equilibrium density gradient centrifugation. Although a molecular weight in excess of 10(7) Da is suggested by gel filtration, the presence of SDS or GuHCl did not alter its elution position, indicating that the large size was not simply due to aggregation. Treatment of this material with disulfide reagents, however, led to a decrease in molecular size. On a relative basis, substantially more of this glycoprotein is present in IPM prepared from retina than from retinal pigment epithelium. While the carbohydrate and amino acid composition are not those of a true 'mucin', the large size and many other properties are quite 'mucin-like'. The carbohydrate composition suggests the presence of both N- and O-glycosidically linked sugar chains. The presence of a mucin-type O-glycosidic linkage is indicated by its susceptibility to alkaline cleavage, with concomitant loss of serine and threonine and increase in 240 nm absorbance; production of a fluorescent product upon reaction with cyanoacetamide; lectin binding properties; and production of N-acetylgalactosaminitol upon alkaline borohydride elimination. This glycoprotein was digested by pronase and trypsin, confirming its protein nature, but was resistant to digestion with chondroitin ABC lyase, hyaluronidase and heparinase, as well as RNAase, indicating that these components were not present to any appreciable extent. ELISA for cartilage keratan sulfate was also negative. Centrifugation in CsCl/GuHCl gradients indicated a density much lower than that of a proteoglycan or nucleic acid as well. In vitro biosynthetic studies suggest that both retina and retinal pigment epithelium may be major sources of material in the IPM. The elution patterns of radioactivity were strikingly similar to the UV elution patterns of IPM. The medium from retinal incubations contained very high molecular weight material which was resistant to enzymes which hydrolyse glycosaminoglycans, suggesting that retina may be the source of this high molecular weight, mucin-like glycoprotein.
通过对新鲜牛眼的光感受器间基质（IPM）进行凝胶过滤，分离出一种分子量非常高的黏蛋白样糖蛋白，并通过氯化铯/盐酸胍（GuHCl）平衡密度梯度离心法将其纯化至表观均一性。尽管凝胶过滤表明其分子量超过10^7 Da，但SDS或GuHCl的存在并未改变其洗脱位置，这表明其大尺寸并非仅仅由于聚集所致。然而，用二硫键试剂处理该物质会导致分子尺寸减小。相对而言，从视网膜制备的IPM中这种糖蛋白的含量比从视网膜色素上皮制备的IPM中要多得多。虽然其碳水化合物和氨基酸组成并非真正“黏蛋白”的组成，但大尺寸和许多其他特性却相当“黏蛋白样”。碳水化合物组成表明存在N-糖苷键和O-糖苷键连接的糖链。其对碱性裂解敏感，同时丝氨酸和苏氨酸损失，240 nm吸光度增加；与氰基乙酰胺反应产生荧光产物；凝集素结合特性；以及碱性硼氢化钠消除后产生N-乙酰半乳糖胺醇，这些都表明存在黏蛋白型O-糖苷键连接。该糖蛋白被链霉蛋白酶和胰蛋白酶消化，证实了其蛋白质性质，但对软骨素ABC裂解酶、透明质酸酶、肝素酶以及RNA酶的消化具有抗性，这表明这些成分在其中的含量并不显著。软骨硫酸角质素的ELISA检测也为阴性。在CsCl/GuHCl梯度中离心表明其密度也远低于蛋白聚糖或核酸。体外生物合成研究表明，视网膜和视网膜色素上皮可能都是IPM中该物质的主要来源。放射性的洗脱模式与IPM的紫外洗脱模式惊人地相似。视网膜孵育培养基中含有分子量非常高的物质，该物质对水解糖胺聚糖的酶具有抗性，这表明视网膜可能是这种高分子量黏蛋白样糖蛋白的来源。
