Nualart F, Hein S, Yulis C R, Zárraga A M, Araya A, Rodríguez E M
Instituto de Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile.
Cell Tissue Res. 1998 May;292(2):239-50. doi: 10.1007/s004410051055.
The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.
连合下器官的大部分分泌物由糖蛋白构成，这些糖蛋白似乎源自540 kDa和320 kDa的两种前体形式。释放到脑室后，这些糖蛋白聚集形成赖氏纤维。我们报告了通过使用抗赖氏纤维血清进行免疫筛选，从牛连合下器官RNA制备的cDNA文库中分离出三个cDNA克隆。通过聚合酶链反应扩增出0.7、1.2和2.5 kb的插入片段，亚克隆到pUC18载体中并进行测序。尽管对这三个插入片段的限制性图谱分析最初表明它们都源自同一mRNA，但序列分析显示，与1.2 kb和2.5 kb的插入片段相比，0.7 kb的插入片段中存在一个短的非同源区域，这表明它们对应于两种不同但高度同源的mRNA。Northern分析显示，连合下器官中存在一种约9.5 kb的单一mRNA，而脉络丛、大脑皮层和肝脏中则没有。原位杂交证实，RNA的表达仅限于牛连合下器官的细胞。针对一种合成肽产生的多克隆抗体，其氨基酸序列是从2.5 kb的cDNA推导出来的，该抗体与牛和大鼠连合下器官-赖氏纤维复合体发生特异性反应。在牛连合下器官的免疫印迹中，该抗体显示出540 kDa的前体形式及其推测的450 kDa加工形式。结论是，克隆的cDNA编码赖氏纤维的主要组成性糖蛋白，在此命名为RF-Gly I。RF-Gly I的测序区域与血管性血友病因子的某些区域和某些粘蛋白显示出高度同源性；它还显示出与spondin家族蛋白质和其他蛋白质中存在的重复序列同源的两个基序。RF-Gly I重复序列的核心序列表明该分子具有蛋白质结合特性。
