Pierce A, Lyon M, Hampson I N, Cowling G J, Gallagher J T
Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom.
J Biol Chem. 1992 Feb 25;267(6):3894-900.
We have used an antiserum raised against a purified heparan sulfate proteoglycan (HSPG) preparation isolated from rat liver to screen a lambda gt11 expression library and have obtained overlapping cDNA clones that contain the full-length coding sequence of an HSPG core protein capable of spanning the plasma membrane. The open reading frame of the rat cDNA encodes a protein of 211 amino acids. The predicted protein sequence (23 kDa) has a high degree of homology with the published partial sequence of the human lung fibroblast HSPG, fibroglycan. The deduced protein sequence contains a 24-amino acid transmembrane domain and a 33-amino acid cytoplasmic domain, both of which are identical with the corresponding regions of human fibroglycan and are highly homologous to the human, hamster, and mouse epithelial HSPG, syndecan. The putative ectodomain, which has 85% homology to fibroglycan, contains three possible glycosaminoglycan attachment sites that may be occupied by heparan sulfate chains. The major 49-kDa core protein in the liver HSPG preparation was found to be reactive to an antibody that specifically recognizes the cytoplasmic domain of fibroglycan. We have used the full-length cDNA clone to analyze the expression of this transmembrane core protein gene in whole tissues and several epithelial and fibroblastoid cell lines. It hybridizes to three mRNA species in all cell and tissue types examined, but in liver, isolated hepatocytes, and kidney, an additional 0.8-kilobase mRNA was detected. The three common messages arise from differential use of alternative polyadenylation sites, whereas the fourth tissue-restricted RNA species represents a related gene transcript. The rat equivalent of human fibroglycan therefore appears to be the major transmembrane proteoglycan in liver, and its widespread expression in many diverse tissues and cells suggests that it plays an important role in cellular interactions.
我们使用了一种针对从大鼠肝脏中分离出的纯化硫酸乙酰肝素蛋白聚糖（HSPG）制剂产生的抗血清，来筛选λgt11表达文库，并获得了重叠的cDNA克隆，这些克隆包含一个能够跨越质膜的HSPG核心蛋白的全长编码序列。大鼠cDNA的开放阅读框编码一个211个氨基酸的蛋白质。预测的蛋白质序列（23 kDa）与已发表的人肺成纤维细胞HSPG纤维糖蛋白的部分序列具有高度同源性。推导的蛋白质序列包含一个24个氨基酸的跨膜结构域和一个33个氨基酸的胞质结构域，这两个结构域与人纤维糖蛋白的相应区域相同，并且与人、仓鼠和小鼠上皮HSPG多配体蛋白聚糖高度同源。推定的胞外结构域与纤维糖蛋白有85%的同源性，包含三个可能被硫酸乙酰肝素链占据的糖胺聚糖附着位点。发现肝脏HSPG制剂中的主要49 kDa核心蛋白与一种特异性识别纤维糖蛋白胞质结构域的抗体发生反应。我们使用全长cDNA克隆来分析这种跨膜核心蛋白基因在全组织以及几种上皮和类成纤维细胞系中的表达。在所检测的所有细胞和组织类型中，它与三种mRNA种类杂交，但在肝脏、分离的肝细胞和肾脏中，检测到另外一种0.8千碱基的mRNA。这三种常见的信使RNA来自于对可变聚腺苷酸化位点的不同使用，而第四种组织限制性RNA种类代表一个相关的基因转录本。因此，大鼠中与人纤维糖蛋白等效的蛋白似乎是肝脏中的主要跨膜蛋白聚糖，并且它在许多不同组织和细胞中的广泛表达表明它在细胞相互作用中起重要作用。
