Beta-ig. Molecular cloning and in situ hybridization in corneal tissues.
Beta-ig. Molecular cloning and in situ hybridization in corneal tissues.
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发表时间：
1997-04
影响因子：
4.4
通讯作者：
Ian Rawe;Q. Zhan;Robert Burrows;Kelly Bennett;Charles Cintron
中科院分区：
文献类型：
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作者：
Ian Rawe;Q. Zhan;Robert Burrows;Kelly Bennett;Charles Cintron
目的利用原位杂交技术，鉴定一种能与兔角膜VI型胶原蛋白结合的蛋白，并确定其在兔角膜组织中的细胞来源。方法采用尿素法提取角膜VI型胶原，经硫酸铵沉淀和凝胶层析纯化。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测胶原蛋白纯度。在用巯基乙醇或二硫苏糖醇还原时，VI型胶原蛋白的α链进入凝胶。除了VI型胶原多肽外，还出现了一条额外的68-kDa蛋白带，表明该蛋白在还原前作为大分子量组分存在。氨基酸测序结果表明，该蛋白与来自人类的β - ig-h3有关。Western blot分析确定其与人蛋白的免疫相似性。用人β - ig-h3 cDNA探针筛选兔间质细胞cDNA文库。阳性克隆测序并进行序列同源性分析。兔cDNA序列制备的寡核苷酸探针用于角膜组织的Northern blot分析和原位杂交。结果SDS-PAGE电泳和68-kDa带前10个n端氨基酸的氨基酸序列分析显示，该蛋白与人腺癌细胞产生的已知蛋白β eg -h3同源性为100%。Western blot分析表明，该68-kDa蛋白在免疫学上与β ig-h3相同。兔cDNA克隆包含完整的编码区，与人β ig-h3和小鼠β ig-m3具有较高的同源性。所得氨基酸序列与这些物种的同源性为92%。兔cDNA序列的寡核苷酸探针检测到基质细胞培养物中与人β - ig-h3 mRNA大小一致的单带mRNA。作者将兔形式的β ig-h3称为β ig，因为该蛋白是从正常兔角膜中获得的，其mRNA来自兔基质细胞的原代培养，而不是克隆细胞系。兔角膜组织原位杂交表明，β - ig mRNA主要存在于正常成人角膜上皮、胎儿间质细胞以及角膜创面愈合中的内皮细胞和间质来源细胞中。正常成人内皮和间质未显示β - ig mRNA标记。结论人、小鼠和家兔蛋白之间高度保守的氨基酸序列同源性以及β - ig信息在角膜愈合和发育过程中的时间表达表明该蛋白在角膜组织的形态发生中起重要作用。
PURPOSE To identify a protein that copurifies with type VI collagen from rabbit cornea and to determine its cell source in rabbit corneal tissues by in situ hybridization. METHODS Type VI collagen was extracted from cornea with urea and purified by ammonium sulfate precipitation and gel chromatography. The purity of the collagen was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction with mercaptoethanol or dithiothreitol, the alpha chains of type VI collagen ran into the gel. In addition to the type VI collagen polypeptides, an extra 68-kDa protein band appeared, suggesting that this protein is present as a large molecular weight component before reduction. Amino acid sequencing indicated that protein was related to beta ig-h3 from humans. Western blot analysis was used to determine immunologic similarity to this human protein. A rabbit stromal cell cDNA library was screened with human beta ig-h3 cDNA probe. Positive clones were sequenced and analyzed for sequence homology. Oligonucleotide probes prepared from rabbit cDNA sequences were used for Northern blot analysis and in situ hybridization of corneal tissues. RESULTS Electroblotting of the SDS-PAGE and amino acid sequence analysis of the first 10 N-terminal amino acids of the 68-kDa band gave 100% homology with a known protein produced by human adenocarcinoma cells, beta ig-h3. This 68-kDa protein was identical immunologically to beta ig-h3 by Western blot analysis. Sequence analysis of a rabbit cDNA clone contained the whole coding region and had high identity with both human beta ig-h3 and mouse beta ig-m3. The deduced amino acid sequence had 92% identity with these species. An oligonucleotide probe from the rabbit cDNA sequence detected a single band of mRNA from cultures of stromal cells consistent in size with human beta ig-h3 mRNA. The authors refer to the rabbit form of beta ig-h3 as beta ig because the protein was obtained from normal rabbit cornea and the mRNA comes from primary cultures of rabbit stromal cells and not from a cloned cell line. In situ hybridization of rabbit corneal tissue indicated that the beta ig mRNA is located primarily in the epithelium of normal adult cornea, in fetal stromal cells, and both endothelium- and stroma-derived cells in healing corneal wounds. Normal adult endothelium and stroma did not show beta ig mRNA label. CONCLUSIONS The highly conserved amino acid sequence homology between the human, mouse, and rabbit proteins and the temporal expression of beta ig message during corneal healing and development suggest this protein plays an important role in the morphogenesis of corneal tissues.