Relation of structure and function of the neural cell adhesion molecule L1 / 基础医学与临床

ABSTRACT

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 pETL1 mutants of several extracellular domains of(L1) were constructed by inserting L1 and its mutants cDNA into plasmid pET-28a(+) 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 cells 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 soobvious 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.