Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation.
Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation.
复制标题
参与海胆胚胎骨架形成的三种初级间充质细胞特异性糖蛋白常见的翻译后修饰的表征。
DOI：
10.1016/0012-1606(92)90243-a
复制
发表时间：
1992
影响因子：
2.7
通讯作者：
Lennarz,WJ
中科院分区：
文献类型：
--
作者：
Kabakoff,B;Hwang,SP;Lennarz,WJ
先前的研究已经确定了一个复杂的n -连接低聚糖链的重要性，该链被单克隆抗体（mAb 1223）识别，在海胆胚胎骨骼成分的形成中被称为针状体。为了进一步研究该表位的功能，在细刺形成前将mAb 1223添加到原代间充质（PM）细胞培养中。该抗体不抑制细胞迁移、细胞附着或针状体沉积的丝状网络的合成。然而，它确实阻断了矿化caco3沿着这些丝状足的沉积，有力地支持了之前提出的1223表位在钙积累和/或沉积中的作用。此前，1223表位与间质特异性蛋白130 kDa （msp 130）相关的研究最为广泛。现在，通过使用mAb 1223对整个胚胎和PM细胞提取物进行Western blot分析，已经确定了另外两个205和250 kDa的蛋白含有1223表位。对这些糖蛋白的发育表达谱的研究表明，这三种糖蛋白都是在针尖形成之前首先表达的，这与这些糖蛋白在这一过程中的作用是一致的。此外，这三种蛋白质都含有乙醇胺、肉豆蔻酸盐和棕榈酸盐，这是糖基磷脂酰肌醇（GPI）锚定的前体。进一步的标记研究揭示了三种蛋白质中GPI锚点的代谢不稳定性的差异；脉冲追踪研究表明，乙醇胺部分在msp 130中是稳定的，但在205-kDa蛋白中迅速被追赶（t12 = 14小时）。磷脂酰肌醇特异性磷脂酶C从PM细胞表面部分释放（50%）msp 130，而对205和250 kda蛋白的释放没有影响。用35 so4标记和PNGase F处理的研究直接证实，这三种蛋白都被硫酸化了，而且大多数硫酸都附着在n -连接的低聚糖链上。因此，PM细胞中的三种主要的mAb 1223反应性糖蛋白也是同时含有硫酸化n -连接低聚糖链和GPI锚点的三种主要蛋白。进一步研究这种有趣的相关性可能有助于确定1223表位在针状体形成过程中的精确功能
Previous studies have established the importance of a complex, N-linked oligosaccharide chain, recognized by a monoclonal antibody (mAb 1223), in the formation of sea urchin embryonic skeletal components known as spicules. To further investigate the function of this epitope, mAb 1223 was added to primary mesenchyme (PM) cell cultures prior to spiculogenesis. The antibody did not inhibit cell migration, cell attachment, or synthesis of the filapodial networks upon which the spicules are deposited. However, it did block deposition of mineralized CaCO 3 along these filapodia, strongly supporting the previously proposed role for the 1223 epitope in calcium accumulation and/or deposition. Previously the 1223 epitope has been most extensively studied in association with a mesenchyme-specific protein of 130 kDa (msp 130). It has now been established, by Western blot analysis of whole embryo and PM cell extracts using mAb 1223, that two other proteins of 205 and 250 kDa contain the 1223 epitope. A study of the developmental profiles of expression of these glycoproteins revealed that all three were first expressed just prior to spiculogenesis, consistent with a role for any or all of these proteins in this process. Additionally all three proteins incorporated ethanolamine, myristate, and palmitate, the precursors of the glycosylphosphatidylinositol (GPI) anchor. Further labeling studies revealed differences in the metabolic lability of the GPI anchor in the three proteins; pulse-chase studies demonstrated that the ethanolamine moiety was stable in msp 130, but was rapidly chased from the 205-kDa protein (T 1 2= 14 hr). Phosphatidylinositol-specific phospholipase C partially released (50%) msp 130 from the PM cell surface, whereas it had no effect on release of the 205-and 250-kDa proteins. Studies with 35 SO 4 labeling and PNGase F treatment directly established that all three proteins are sulfated, and that most of the sulfate is attached to the N-linked oligosaccharide chains. Thus, the three major mAb 1223-reactive glycoproteins in PM cells are also the three major proteins containing both sulfated N-linked oligosaccharide chains and GPI anchors. Further investigation of this intriguing correlation may help to define the precise function of the 1223 epitope in the process of spicule formation