The 67-kDa enzymatically inactive alternatively spliced variant of β-galactosidase is identical to the elastin/laminin-binding protein
The 67-kDa enzymatically inactive alternatively spliced variant of β-galactosidase is identical to the elastin/laminin-binding protein
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DOI：
10.1074/jbc.273.11.6319
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发表时间：
1998-03-13
影响因子：
4.8
通讯作者：
Hinek, A
中科院分区：
文献类型：
--
作者：
Privitera, S;Prody, CA;Hinek, A
我们以前的研究表明，免疫学和功能的相似性，以及部分序列同源性，之间的酶活性的选择性剪接变体的人β-半乳糖苷酶（S-gal）和67 kDa的弹性蛋白/层粘连蛋白结合蛋白（EBP）从羊。为了进一步确定EBP的遗传起源，构建了全长人S-gal cDNA克隆并进行体外转录/翻译。将该cDNA转染COS-1细胞和EBP缺陷的绵羊动脉导管平滑肌细胞（SMC）。在体外翻译产生了非糖基化形式的S-gal蛋白，其与抗β-半乳糖苷酶抗体发生免疫反应，并与弹性蛋白和层粘连蛋白亲和柱结合。S-gal cDNA转染COS-1和DA SMC增加了67 kDa蛋白的表达，该蛋白免疫定位于细胞内和细胞表面，当从细胞中提取时，与弹性蛋白结合。S-gal转染的细胞显示出对弹性蛋白覆盖的培养皿的粘附性增加，这与新产生的S-gal编码蛋白的细胞表面分布一致。DA SMC的转染另外校正了它们受损的弹性纤维组装。这些结果最终确定β-半乳糖苷酶的67-kDa剪接变体为EBP。
Our previous studies showed immunological and functional similarities, as well as partial sequence homology, between the enzymatically inactive alternatively spliced variant of human beta-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding protein (EBP) from sheep. To define the genetic origin of the EBP further, a full-length human S-gal cDNA clone was constructed and subjected to in vitro transcription/translation. The cDNA was also transfected into COS-1 cells and into the EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus (DA). In vitro translation yielded an unglycosylated form of the S-gal protein, which immunoreacted with anti-beta-galactosidase antibodies and bound to elastin and laminin affinity columns. S-gal cDNA transfections into COS-1 and DA SMC increased expression of a 67-kDa protein that immunolocalized intracellularly and to the cell surface and, when extracted from the cells, bound to elastin. The S-gal-transfected cells displayed increased adherence to elastin-covered dishes, consistent with the cell surface distribution of the newly produced S-gal-encoded protein. Transfection of DA SMC additionally corrected their impaired elastic fiber assembly. These results conclusively identify the 67-kDa splice variant of beta-galactosidase as EBP.