Neuroglobin mutation associated with hypoxia adaptation in Tibet chicken

Globin protein family plays an important role in storing and transporting oxygen. As a newly reported globin, the revealed function of neuroglobin includes binding and storing oxygen as well as facilitating the utilization of oxygen in neuronal cells. In the present study, mutations in exons of chicken Ngb gene were identified with the method of sequencing and created restriction site PCR in Tibet chicken and other four lowland chicken breeds. The mutations of Lys-2224(E4)-Asn and Ser-2279(E4)-Gly were identified in exon 4 of the gene. The Lys-2224(E4)-Asn mutation existed only in Tibet chicken and the mutation frequencies increased with increasing altitude. Analysis of the haplotype and diplotype of the two mutations in Tibet chicken populations of different altitudes showed that the frequencies of TG haplotype and TTGG diplotype also increased with increasing altitude, while the reverse tendency was found on GGAA diplotype. Under the hypoxic simulation incubation, the main haplotype was TG in living embryos and GA in dead embryos. The results showed that the Lys-2224(E4)-Asn mutation may be a specific mutation associated with hypoxia adaptation in Tibet chicken.     

Neuroglobin mutation associated with hypoxia adaptation in Tibet chicken

Globin protein family plays an important role in storing and transporting oxygen. As a newly reported globin, the revealed function of neuroglobin includes binding and storing oxygen as well as facilitating the utilization of oxygen in neuronal cells. In the present study, mutations in exons of chicken Ngb gene were identified with the method of sequencing and created restriction site PCR in Tibet chicken and other four lowland chicken breeds. The mutations of Lys-2224(E4)-Asn and Ser-2279(E4)-Gly were identified in exon 4 of the gene. The Lys-2224(E4)-Asn mutation existed only in Tibet chicken and the mutation frequencies increased with increasing altitude. Analysis of the haplotype and diplotype of the two mutations in Tibet chicken populations of different altitudes showed that the frequencies of TG haplotype and TTGG diplotype also increased with increasing altitude, while the reverse tendency was found on GGAA diplotype. Under the hypoxic simulation incubation, the main haplotype was TG in living embryos and GA in dead embryos. The results showed that the Lys-2224(E4)-Asn mutation may be a specific mutation associated with hypoxia adaptation in Tibet chicken.     

Expression,purification and spectra characterization of neuroglobin

The expression, purification and spectra characterization of recombinant human neuroglobin （NGB） are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 （DE3） plys cells and expressed in TB culture medium. The results indicated that the expression amount of NGB is about 10 percent of the total protein in cells. The NGB protein was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and a Hiprep 16/10 Q FF anion exchange column, and a red soluble protein was obtained which showed a single band in electrophoresis. Electrospray ionization mass spectrometry （ESI-MS） showed that its molecular weight is 16930.0 Da. UV-spectra indicated that the reduced NGB has a strong absorption peak at 425 nm, and two weak peaks at 531 and 559 nm, which can be assigned to γ, β and α bands of porphyrin, respectively, and the oxidized NGB has a strong absorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maximal excitation wavelength is at 281 nm and its maximal emission wavelength is at 338 nm. CD spectra indicated that its secondary structure is a typical α helix, and has a positive peak at 410 nm induced by heme, The NGB protein is stable when the pH is higher than 4.     

中国人神经珠蛋白(NGB)基因克隆与序列分析

应用RT-PCR方法从中国人胎脑组织中扩增出神经珠蛋白(NGB)特异性编码基因片段,并将其克隆到pMD18-TVector中,构建了pMD18-T-hNGB克隆质粒,然后进行测序分析并与国外报道的NGB序列相比较.结果表明,本文获得的中国人NGB编码基因序列与国外序列的同源性为98%.     

人工神经红蛋白的亚硝酸盐还原酶活性研究

为更好地探究神经红蛋白血红素铁配位情况改变对亚硝酸盐还原酶活性的影响,以神经红蛋白作为血红素蛋白模型分子,在其远端引入甲硫氨酸。通过紫外-可见光分光光度计动力学以及电子顺磁共振波谱等方法研究,其亚硝酸盐还原酶活性的二级速率常数k_nitrite从0.44 L/(mol·s)提高到487 L/(mol·s)。结果表明,血红素口袋结构对蛋白的亚硝酸盐还原酶活性有较好的调节作用。     

Circular dichroism spectral studies on the recombinant human neuroglobin

Neuroglobin (NGB) is a newly discov- ered member of the hemoglobin superfamily that is primarily expressed in the brain of humans and other vertebrates. The effects of protein concentration, solvent, pH and temperature on the secondary structure of NGB were investigated by employing far UV circular dichroism (CD) spectroscopy. The results show that NGB exists mainly in α-helix form when its concentration is less than 10 μmol/L. However, its α-helix content decreases with the increase of con- centration in the range of 10―40 μmol/L and remains unchanged when the concentration is higher than 40 μmol/L, which suggest that NGBs form intermolecular disulfide bond and aggregate in higher concentration. The α-helix content of NGB in methanol and ethanol is a little higher than that in water, indicating a higher stability of NGB in these solvents. NGB loses its α-helical secondary structure in either acidic or alka- line solution to some extent. Although increased temperature destabilizes the α-helices of NGB, over 16% of α-helices can be kept at 110°C. Therefore, NGB is a protein with hyperthermal stability.