Muragaki Y, Shiota C, Inoue M, Ooshima A, Olsen B R, Ninomiya Y
Department of Pathology, Wakayama Medical College, Japan.
Eur J Biochem. 1992 Aug 1;207(3):895-902. doi: 10.1111/j.1432-1033.1992.tb17122.x.
Type-VIII collagen is a major constituent of Descemet's membrane and contains two genetically distinct alpha chains, alpha 1(VIII) and alpha 2(VIII). We have previously cloned the human alpha 1(VIII) and alpha 2(VIII) genes and determined that they are located on chromosomes 3 and 1, respectively. Comparison of the alpha 1(VIII) and alpha 2(VIII) genes with the alpha 1(X)-collagen gene showed that the structure of the three genes and the sequence of their collagen polypeptides were strikingly similar. Therefore, we have grouped the three genes in a common subclass of collagens which we have named the short-chain collagens because of the relatively small size of their triple-helical domains. In the present study, we have isolated and characterized a mouse gene fragment encoding the entire mouse alpha 1(VIII)-collagen chain and determined the complete primary structure of the polypeptide chain. The size of mouse alpha 1(VIII)-collagen mRNA, as estimated by Northern-blot analysis, was 4.2 kb, compared with 2.8 kb previously reported for the corresponding rabbit mRNA. By cloning and sequencing of four overlapping cDNA, we demonstrate that this larger size of mouse alpha 1(VIII) mRNA is due to a larger 3' untranslated region in the mouse transcript. Using the gene fragment as a probe, we performed Northern-blot hybridization analysis of RNA prepared from newborn mice and demonstrated that alpha 1(VIII) collagen mRNA is expressed at high levels in the calvarium, eye and skin. In situ hybridization revealed that alpha 1(VIII) RNA is present in skin keratinocytes, corneal epithelial and endothelial cells, lens epithelial cells, as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.
VIII型胶原是Descemet膜的主要成分,由两条基因上不同的α链,即α1(VIII)和α2(VIII)组成。我们之前已克隆了人α1(VIII)和α2(VIII)基因,并确定它们分别位于3号和1号染色体上。将α1(VIII)和α2(VIII)基因与α1(X)-胶原基因进行比较,结果显示这三个基因的结构及其胶原多肽的序列极为相似。因此,我们将这三个基因归为胶原的一个共同亚类,因其三螺旋结构域相对较小,我们将其命名为短链胶原。在本研究中,我们分离并鉴定了一个编码完整小鼠α1(VIII)-胶原链的小鼠基因片段,并确定了该多肽链的完整一级结构。通过Northern印迹分析估计,小鼠α1(VIII)-胶原mRNA的大小为4.2 kb,而之前报道的相应兔mRNA大小为2.8 kb。通过对四个重叠cDNA的克隆和测序,我们证明小鼠α1(VIII) mRNA较大是由于小鼠转录本中3'非翻译区较大。使用该基因片段作为探针,我们对新生小鼠制备的RNA进行了Northern印迹杂交分析,结果表明α1(VIII)胶原mRNA在颅盖骨、眼睛和皮肤中高水平表达。原位杂交显示,α1(VIII) RNA存在于皮肤角质形成细胞、角膜上皮和内皮细胞、晶状体上皮细胞,以及软骨和颅盖骨周围的间充质细胞和脑周围的脑膜中。