Functional Analysis of extracellular matrix tenascin-X

为了确定细胞外基质腱生蛋白-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、肝和肾中纯化了约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率和最终数量。此外，糖生化特性的TNX进行了表征凝集素印迹分析。凝集素印迹显示TNX中存在N-聚糖。此外，根据对PNA的反应，肝和肾的TNX也具有O-聚糖。与AAL的结合表明，来自三种组织的TNX具有与五糖核心连接的α 1，6岩藻糖。对SSA而不是对MAM的反应表明TNX中存在与半乳糖连接的唾液酸α 2，6。在从各种器官中纯化细胞TNX的过程中，我们在小鼠中鉴定了分子量为200 kDa的血清型TNX（sTNX）。200-kDa sTNX含有15个纤连蛋白III型样（FNIII）重复序列和一个与细胞TNX C-末端部分相同的纤维蛋白原结构域。通过脾匀浆对细胞TNX的蛋白水解裂解产生sTNX，随后检测L、MCF 7或H1299细胞对TNX的粘附。L细胞不粘附于TNX，而MCF 7或H1299细胞仅粘附于其上，但大多数粘附的细胞保持圆形或最小程度地铺展而不形成显著的层状结构。此外，将MCF 7细胞铺在用TNX包被在纤连蛋白（FN）上的组织培养塑料上。TNX对FN促进的细胞贴壁和铺展有轻度抑制作用。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.