The Molecular Basis Of Neuroglobin And Its Significance In Protecting Brain From Hypoxic Injure

Neuroglobin (NGB) is a newly discovered O2-binding protein specifically existing in nervous system. It has been proved that NGB could protect the neuron from hypoxic injury. As NGB is a hexacoordinated globin, its conformational changes occurr upon O2 association or dissociation, and even its interaction with other proteins may be affected. So NGB may function as 62 sensor, triggering a signal transduction pathway that modulates the activity of hypoxia related proteins and protects the neuron. In order to explore the molecular basis of neuroglobin, the pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by yeast two hybrid system. The amino acid sequence encoded by one of the clones interacting with neuroglobin was the C terminal of Na*, K+-ATPase beta 2 subunit (NKAlb2). The first fifteen amino acids of NGB from N terminal were found to be essential for the interaction between NGB and NKAlb2 by yeast two hybrid experiments with a serials of NGB truncated mutants. Then the full length cDNA sequence of coding region for NKAlb2 was obtained from human fetal brain cDNA library by PCR. A set of experiments were designed to test the interaction between NGB and NKAlb2. The interaction of NGB and NKAlb2 was confirmed at first by GST- pull down in vitro. And the interaction was proved by co-immunoprecipitation test in vivo. Moreover, NGBand NKAlb2 were co-localized in neuron by indirect fluorescent immunocytochemistry. The results also showed that Na+,K+-ATPase activity could be inhibited by NGB in vitro in a dose-dependent model. NGB was up-regulated one to two times under the hypoxic condition. Moreover NGB and NKAlb2 also co-localized in neuron under the hypoxia, while the expression of NKAlb2 couldn't be regulated. About ten percent of Na+,K+-ATPase activity was inhibited, which was calculted as the dose-bependent model. Since Na+,K+-ATPase is one of the main ATP consuming enzyme in brain, slight inhibition on Na+,K*-ATPase activity could conserve the energy and reduce the demand for oxygen as well as protect the neuron in early stage of hypoxia. It is suggested thathypoxia-induced NGB could inhibit the Na+,K+-ATPase activity to certain extent to protect the neuron from hypoxic injury.