Functional Analysis of extracellular matrix tenascin-X

为了确定细胞外基质tenascin-X （TNX）在胶原纤维形成中的作用，我们首先开发了一种简单快速的方法从小鼠各种组织中分离TNX。我们从脾脏、肝脏和肾脏中纯化了大约350 kda的细胞间质TNX。接下来，我们研究了TNX对I型胶原原纤维形成的影响。因此，我们发现来自这些组织的TNX增加了原纤维形成的速度和最终数量。此外，通过凝集素印迹分析表征TNX的糖生化特性。凝集素印迹显示TNX中存在n -聚糖。此外，来自肝脏和肾脏的TNX也具有基于对PNA反应的o -聚糖。与AAL的结合表明来自三种组织的TNX具有与aa1,6连接到五糖核心的病灶。对SSA的反应而不是对MAM的反应表明TNX中存在连接a2,6和半乳糖的唾液酸。在从各种器官中纯化细胞TNX的过程中，我们在小鼠中鉴定出了分子质量为200 kDa的TNX （sTNX）血清形式。200 kda的sTNX包含15个纤维连接蛋白iii型（FNIII）重复序列和一个与细胞TNX的c端部分相同的纤维蛋白原结构域。sTNX是通过脾脏匀浆对细胞TNX进行蛋白水解裂解而产生的。随后，检测L、MCF7或H1299细胞对TNX的粘附性。L细胞不粘附TNX， MCF7和H1299细胞粘附较少，但大多数粘附细胞呈圆形或扩散，未形成明显的片层。将MCF7细胞置于组织培养物上，用TNX包被纤维连接蛋白（FN）。发现TNX对FN促进的细胞附着和扩散有轻度抑制作用。这些结果表明TNX具有调节细胞-细胞外基质相互作用的能力。

In order to determine the roles of extracellular matrix tenascin-X (TNX) in collagen fibrillogenesis, we first developed an easy and fast method to isolate TNX from various tissues in mice. We purified approximately 350-kDa cellular interstitial TNX from spleen, liver and kidney. Next, we investigated the effects of TNX on type I collagen fibril formation. Consequently, we found that TNX from these tissues increased the rate and the final quantity of fibril formation. Furthermore, glycobiochemical properties of TNX were characterized by lectin blot analysis. Lectin blots revealed the presence of N-glycan in TNX. In addition, the TNX from liver and kidney also possessed O-glycan based on the reaction to PNA. The binding to AAL indicated that TNX from the three tissues possesses fucose linked a1,6 to a pentasaccharide core. The reaction to SSA but not to MAM suggested the presence of sialic acid linked a2,6 to galactose in TNX. During the purification of cellular TNX from various organs, we identified the serum form of TNX (sTNX) with a molecular mass of 200 kDa in mouse. The 200-kDa sTNX contains 15 fibronectin type III-like (FNIII) repeats and a fibrinogen domain identical to the C-terminal portion of cellular TNX. sTNX was generated by proteolytic cleavage of cellular TNX by spleen homogenate.Subsequently, L, MCF7 or H1299 cells were tested for adhesion to TNX. L cells did not adhere to TNX, whereas MCF7 or H1299 cells adhered to it just a few, but most of the adhered cells remained rounded or spread minimally without forming significant lamellae. Furthermore, MCF7 cells were plated on tissue culture plastic coated on fibronectin (FN) with TNX. TNX was found to have mild inhibitory effects on the cell attachment and spreading promoted by FN. These results indicated that TNX has its ability to modulate cell-extracellular matrix interactions.