神经细胞黏附因子L1结构与功能的关系

Relation of structure and function of the neural cell adhesion molecule L1

目的获得重组神经细胞黏附因子L1(L1),研究其结构与功能的关系。方法通过RT-PCR方法,扩增出L1cDNA;构建原核表达pET28a+和真核表达pcDNA3.0载体;并构建L1胞外域不同结构基序的原核表达载体;将pET L1s转化大肠杆菌BL21(DE3),获得表达菌株BLL1s;将pcDNA3.0-L1真核表达质粒转染入PC12细胞中,筛选稳定表达L1的基因工程细胞株PC12-L;用原核表达的L1胞外不同结构域蛋白刺激PC12L1基因工程细胞,观察其分化情况。结果IgI-FN5胞外域I、g(Ⅰ-Ⅵ)免疫球蛋白样结构域和FN(1-5)纤连蛋白型Ⅲ重复序列均可明显促进PC12-L1细胞突起生长,具有生物学活性;Ig(Ⅴ-Ⅵ)也可促进PC12-L1细胞突起生长,但生物活性显著降低;FN(3-5)不能促进PC12-L1细胞突起生长,没有生物活性。结论L1分子胞外至少有2个结构域Ig(Ⅰ-Ⅵ)和FN(1-5)可显著促进PC12-L1细胞的突起生长,对L1分子生物活性的维持必不可少;胞外免疫球蛋白结构域Ig(Ⅴ-Ⅵ)对维持L1分子的生物活性不十分重要;纤连蛋白型Ⅲ重复序列结构域FN(3-5)对维持L1分子的生物活性不重要。

Objective To obtain recombinant neural cell adhension molecule L1 （L1） and to study its structure and biological activity. Methods The cDNA encoding the mature rat L1 was isolated using RT-PCR from total RNA extracted from newborn SD-rat hippocampus tissue. The expression plasmid pET_ L1 mutants of several extracellular domains of L1 were constructed by inserting L1 and its mutants cDNA into plasmid pET-28 a （ ＋ ） containing T7 promoter and transformed into E. coli BL21 （DE3）. A series expression strain BLL1 mutants were selected. Recombinant L1 and its mutant proteins were expressed at levels about 18.5% -30% of total bacteria protein in form of inclusion body after the induction. By Ni^2＋ chelation affinity chromatography, up to 90 % L1＇s mutant proteins were purified. The expressed plasmid pcDNA3-L1 were transfected into PC12 ceils and constructed PC12-engineered cells which stably and highly expressed the L1. Results Purified and refolded IgI-FN5,Ig（ Ⅰ -Ⅵ） and FN（1-5） fragments could significantly promote the neurite outgrowth of PC12-engineered cells;fragment Ig （ Ⅴ -Ⅵ ） also can promote the neurite outgrowth but not so obvious as the before; fragment FN（3-5） have no function to induce the neurite outgrowth of PC12-engineered cells. Conclusions These results suggested that there are at least two segments in extracellular domains of L1 Ig （ Ⅰ -Ⅵ ） and FN（1-5） fragments are critical for inducing the neurite outgrowth of PC12-L1 cells and the key amino acids for signaling transduction located in the segments of Ig（ Ⅰ-Ⅵ ） and FN（ 1-5）; fragment Ig（ Ⅴ-Ⅵ ） is necessary but not crucial for promoting the neurite outgrowth; fragment FN（3-5） is not important for L1 biological function.