Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins.
Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins.
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DOI：
10.1016/s0021-9258(18)53809-6
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发表时间：
1993-02
期刊：
影响因子：
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通讯作者：
I. Aukhil;P. Joshi;Y. Yan;H. Erickson
中科院分区：
文献类型：
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作者：
I. Aukhil;P. Joshi;Y. Yan;H. Erickson
我们已经产生了一组细菌表达蛋白对应于10段的腱生蛋白和两个纤连蛋白，并测试它们的肝素结合和细胞粘附。我们使用聚合酶链反应克隆，以终止精确的区段在域边界。肝素结合活性被映射到两个不同的腱蛋白片段：一个包含第四和第五纤连蛋白III型结构域，和TNfbg，纤维蛋白原样末端旋钮。TNfbg也支持原代大鼠胚胎皮肤成纤维细胞的粘附，但其他tanascin片段均不支持。成纤维细胞没有在TNfbg上扩散，而是保持圆形。细胞与TNfbg的结合发生在二价阳离子存在或不存在的情况下，并且不被RGD肽抑制，这表明整合素不参与。成纤维细胞结合TNfbg强烈抑制可溶性肝素，肝素酶处理的细胞，或培养条件下，导致硫酸化不足的蛋白聚糖。这些观察结果表明，细胞附着TNfbg介导的细胞表面蛋白聚糖。我们还在pNUT哺乳动物细胞表达载体中构建了人腱生蛋白的最大和最小剪接变体的全长cDNA构建体，以及第14个表皮生长因子样结构域后截短的cDNA构建体。稳定转染的幼仓鼠肾细胞系分泌大量的腱生蛋白，并将其组装成正常的六臂体，臂长与构建体相对应。
We have produced a set of bacterial expression proteins corresponding to 10 segments of tenascin and two of fibronectin and tested them for heparin binding and cell adhesion. We used polymerase chain reaction cloning to terminate the segments precisely at domain boundaries. Heparin binding activity was mapped to two different tenascin segments: one comprising the fourth and fifth fibronectin type III domains, and to TNfbg, the fibrinogen-like terminal knob. TNfbg, but none of the other tanascin segments, also supported adhesion of primary rat embryo skin fibroblasts. The fibroblasts did not spread on TNfbg but remained rounded. Cell binding to TNfbg occurred in the presence or absence of divalent cations and was not inhibited by RGD peptides, suggesting that integrins are not involved. Fibroblast binding to TNfbg was strongly inhibited by soluble heparin, by treating the cells with heparitinase, or by culture conditions that cause undersulfation of proteoglycans. These observations suggest that cell attachment to TNfbg is mediated by cell surface proteoglycans. We have also made full-length cDNA constructs for the largest and smallest splice variants of human tenascin, as well as one truncated after the 14th epidermal growth factor-like domain, in the pNUT mammalian cell expression vector. Stably transfected baby hamster kidney cell lines secreted large quantities of tenascin, and this was assembled into normal hexabrachions, the arm length corresponding to the construct.