Neurite Guidance Cues Netrin-4 And Nnat Interactions And Expression Correlation

During development of nervous system for establishing neuronal connections, several guidance factors including ephrin, slit, netrin and semaphorin were critically important for mediating accurate guidance of developing axons to right targets. Here we show the structure, function and character of expression of netrin-4, a novel member of netrin family and its interaction with a novel protein neuronatin (NNAT).At first, we prepared the prokaryotic expression vectors of netrin-4 and its three domains (Laminin N-terminal, EGF repeat, Netrin C-terminal). The fusion proteins with GST were successfully induced with IPTG treatment. For studying the function of Netrin-4, we prepared the special antibody of netrin-4 using synthesized peptide. To better understanding the molecular mechanism of netrin-4, the yeast two-hybrid system was used to identify netrin-4 interaction protein from human fetal brain cDNA library. A novel protein, neuronatin (NNAT) was found to have high affinity with netrin-4 and confirmed by GST-pull down test. In addition, we found that the EGF repeat domain was an important structure for netrin-4-NNAT interaction. To clarify the significance of interaction between netrin-4 and NNAT which function was currently unknown, we further checked the sub-cellular localization of NNAT fused with GFP protein. Interestingly, the NNAT protein was found to be located at nucleus. Using flow cytometer, we also found that excessive expression of fusion protein would induce the change of cell cycle. After creating the engineering cell lines (CHO cells) which could steadily express NNAT following routine protocol, we found that NNAT protein still localized at the nucleus and cell cycle was affected by NNAT. These results indicate that NNAT might control the proliferation and differentiation of neuronal cells through its localization in nucleus. We further checked the expression of netrin-4 and Nnat in 11 cell lines and primary cultured neuron using RT-PCR. The results showed that netrin-4 and Nnat could only co-express in neuronal cells. The expression level of netrin-4 mRNA and Nnat mRNA were different during the development of brain. Nnat mRNA...