Midura R J, Toledo O M, Yanagishita M, Hascall V C
Proteoglycan Chemistry Section, National Institute of Dental Research, Bethesda, Maryland 20892.
J Biol Chem. 1989 Jan 25;264(3):1414-22.
Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.
带有巩膜边缘的角膜外植体取自第18天的鸡胚，新鲜制备后在各种放射性前体存在的情况下于体外培养3小时。从基质组织和培养基中分离出放射性标记的蛋白聚糖进行分析。在角膜基质中鉴定出两种主要的蛋白聚糖。一种含有硫酸皮肤素，另一种含有硫酸角质素；每种的结构分析见随附论文（米杜拉，R.J.，和哈斯卡尔，V.C.（1989年）《生物化学杂志》264，1423 - 1430）。在基质提取物中还检测到一种与主要形式不同的少量硫酸角质素蛋白聚糖、少量硫酸乙酰肝素蛋白聚糖和一些硫酸化糖蛋白。硫酸皮肤素蛋白聚糖的生物合成在体外和卵内都是稳定的，而主要的硫酸角质素蛋白聚糖的生物合成仅在卵内稳定。尝试了各种处理方法以在培养过程中随时间维持较高的硫酸角质素合成速率。将角膜外植体冷却至5摄氏度是唯一能在体外显著降低硫酸角质素合成下降的处理方法。在培养基中观察到三种主要的蛋白聚糖。两种是硫酸皮肤素蛋白聚糖，似乎主要来源于角膜外植体周围的巩膜组织。第三种蛋白聚糖含有硫酸角质素。与在基质中发现的硫酸角质素蛋白聚糖相比，它的尺寸较小且电荷密度较低，但两者的核心蛋白大小似乎相似。这种蛋白聚糖似乎是在基质中合成并分泌到培养基中的。在培养基中还检测到少量硫酸乙酰肝素蛋白聚糖和一些硫酸化糖蛋白。
