Gibson M A, Hatzinikolas G, Davis E C, Baker E, Sutherland G R, Mecham R P
Department of Pathology, University of Adelaide, South Australia.
Mol Cell Biol. 1995 Dec;15(12):6932-42. doi: 10.1128/MCB.15.12.6932.
Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.
针对弹性蛋白相关微原纤维的一个组分原纤蛋白1（MP340）的单克隆抗体，被用于筛选由牛项韧带mRNA构建的cDNA文库。挑选出的一个克隆（cL9；1.2 kb）在Northern（RNA）印迹上与项韧带mRNA杂交，产生了两条丰富的mRNA条带，大小分别为9.0 kb和7.5 kb，这两条带与原纤蛋白mRNA（10 kb）明显不同。进一步的文库筛选以及随后通过cDNA末端快速扩增（RACE）技术进行的逆转录PCR，导致分离出了对应于约6.7 kb mRNA的其他重叠cDNA。所编码的蛋白质与最近鉴定出的一种名为潜伏转化生长因子β1（TGF-β1）结合蛋白2（LTBP-2）的人类蛋白质表现出约80%的序列相似性，表明该新蛋白质是牛LTBP-2。牛LTBP-2 cDNA探针在人染色体14q24.3上的特异性定位证实了这一点，人染色体14q24.3正是人类LTBP-2基因的位点。牛LTBP-2的结构域结构与人类LTBP-2非常相似，包含20个6 - 半胱氨酸表皮生长因子样重复序列，其中16个具有钙结合的共有序列，同时还有4个原纤蛋白和LTBP-1特有的8 - 半胱氨酸基序。还存在一个牛LTBP-2特有的4 - 半胱氨酸序列，它与8 - 半胱氨酸基序相似。针对两个独特的牛LTBP-2肽段产生的抗体在组织切片中特异性定位于弹性蛋白相关微原纤维，表明LTBP-2与这些结构紧密相关。免疫印迹实验确定推定的LTBP-2同工型，在培养的弹性组织细胞释放到培养基中的是一种260 kDa的蛋白，在组织提取物中是更大的290 kDa和310 kDa的蛋白。大部分组织来源的LTBP-2需要用6 M胍处理才能溶解，表明该蛋白质与微原纤维紧密结合。大多数胍溶解的LTBP-2似乎是单体，表明它既不参与自身的二硫键结合聚集，也不与潜伏TGF-β发生二硫键结合聚集。额外的LTBP-2对6 M胍溶解有抗性，但很容易用还原性盐溶液提取。这种处理对包括原纤蛋白1和微原纤维相关糖蛋白在内的微原纤维成分的溶解相对特异。因此，可以推断一些LTBP-2通过可还原的二硫键与微原纤维共价结合。证据表明LTBP-2在弹性纤维结构和组装中具有直接作用，这可能与其生长因子结合特性无关。因此，LTBP-2似乎与LTBP-1和原纤蛋白都具有功能特性。
