Hoffmann A, Gath U, Gross G, Lauber J, Getzlaff R, Hellwig S, Galla H J, Conradt H S
Gesellschaft für Biotechnologische Forschung, Department of Molecular Biology, Braunschweig, Germany.
J Cell Physiol. 1996 Nov;169(2):235-41. doi: 10.1002/(SICI)1097-4652(199611)169:2<235::AID-JCP2>3.0.CO;2-P.
Primary porcine choroid plexus epithelial cells cultivated in chemically defined medium maintain their epithelial characteristics and form confluent monolayers. They produce a fluid the composition of which resembles cerebrospinal fluid. The present study demonstrates constitutive secretion of large amounts of beta-trace protein. This intrathecally synthesized protein is a prominent polypeptide constituent of natural cerebrospinal fluid. According to the identity of amino acid sequences it has previously been tentatively identified as a prostaglandin-D synthase and as a member of the lipocalin protein family. beta-Trace was purified from cell culture supernatants and was subjected to tryptic digestion and amino acid sequencing of the resulting peptides. The complete primary structure of the protein was obtained by additional isolation of the cDNA from cultured epithelial cells. The porcine 163-amino acid polypeptide showed 69% identity with the human beta-trace and contained two N-glycosylation sites occupied by complex-type oligosaccharides as is the case for the human protein. The amino acid sequences around the N-glycosylation sites of mammalian beta-trace proteins (porcine, human, murine, and rat) were highly conserved. The nucleotide sequence was found to be less conserved; the porcine cDNA had a strikingly high GC-content (67%). The constitutive secretion of beta-trace protein from the in vitro cultivated porcine choroid plexus epithelial cells demonstrates that the cells have retained their major in vivo physiological properties: secretion of cerebrospinal fluid proteins. Therefore, this in vitro culture system may be used as a versatile tool for studying the regulation of the formation of cerebrospinal fluid.
在化学成分明确的培养基中培养的原代猪脉络丛上皮细胞保持其上皮细胞特征并形成汇合的单层。它们产生一种成分类似于脑脊液的液体。本研究证明了大量β-微量蛋白的组成型分泌。这种鞘内合成的蛋白是天然脑脊液中的一种主要多肽成分。根据氨基酸序列的一致性，它先前已被初步鉴定为前列腺素-D合成酶和脂质运载蛋白家族的一员。从细胞培养上清液中纯化β-微量蛋白，并对所得肽进行胰蛋白酶消化和氨基酸测序。通过从培养的上皮细胞中进一步分离cDNA获得了该蛋白的完整一级结构。猪的163个氨基酸的多肽与人类β-微量蛋白具有69%的一致性，并且含有两个被复合型寡糖占据的N-糖基化位点，人类蛋白也是如此。哺乳动物β-微量蛋白（猪、人、小鼠和大鼠）N-糖基化位点周围的氨基酸序列高度保守。发现核苷酸序列的保守性较低；猪的cDNA具有极高的GC含量（67%）。体外培养的猪脉络丛上皮细胞组成型分泌β-微量蛋白，表明这些细胞保留了其主要的体内生理特性：脑脊液蛋白的分泌。因此，这种体外培养系统可作为研究脑脊液形成调节的通用工具。
