Molecular Mechanisms Of Yak (Bos Grunniens) AQPs In Resistance To High Altitude Cerebral Edema

[Objective]Hypoxia is one of the most important causes of majority diseases, especially brain disease. The high altitude hypoxia seriously hindered the development, exploration, and application of the natural resources in the Qinghai-Tibet Plateau. Due to the particularity of the extreme habitats in the Qinghai Tibet Plateau and the self adaptability of the low elevation species, when the low altitude animal rapidly accessing to the Q inghai Tibet plateau, which leading to the brain metabolism is easily to be disturbed, and further form the high altitude cerebral edema. However, so far, the regulation mechanism of the water and energy metabolism of the plateau animal brain and the mechanism of resistance to the edema are still unknown. Yak (Bos grunniens) is the only large mammal adaption to the extreme hypoxia environment of the Qinghai Tibet Plateau, which plays very important roles in the plateau animal production and plateau adaptation research. In the AQPs, AQP4 and AQP9 are more abundant molecules in the mammalian brain; they not only play a core role in the transportation of water and energy metabolism in the brain cells, but also play an important role in the pathophysiological process of brain edema. Therefore, in this paper, the Qinghai Tibet plateau yak as natural’ hypoxia adaptation model’, the low-altitude cattle as control, we initially study the molecular mechanism of Qinghai Tibet Plateau animals AQPs resistance on the high altitude cerebral edema.[Methods](1) Gene cloning technique; (2) Bioinformatics method; (3) Real-time quantitative PCR; (4) Immunohistochemical method; (5) Based on bioinformatics software and bibliographic database, such as PubMed, CNKI, Google, the gene regulatory network of AQPs-related hypoxia ischemic brain edema is constructed by bioinformatics techniques.[Results](1) The initiation codon of AQP4 is ATG and its termination codon is TGA. The coding region sequence of yak AQP4 contains a complete ORF (966 bp) encoding 323 amino acid (gene ID:KM609432); the initiation codon of AQP9 is ATG and its termination codon is TAA. The coding region sequence of yak AQP9 contains a complete ORF (885 bp) encoding 295 amino acid (gene ID:KM820838).(2) Construction of eukaryotic expression vector of the coding region of yak AQP4 and AQP9.(3) The comparison results analysed by bioinformatics method:It showed the highest sequence homologies of yak AQP4 and AQP9 with those of cattle is 99.8% and 99.3%, respectively. There was one difference of AQP4 amino acid between the yak and cattle. The amino acid of yak AQP4 is Ser and the one of cattle AQP4 is Ala; There were two difference of AQP9 amino acid between the yak and cattle. The 9th amino acid of yak AQP9 is Glu and the one of cattle AQP9 is Lys. And the 273th amino acid of yak AQP9 is Leu and the one of cattle AQP9 is Pro; Though the second structures of AQP4 and AQP9 are mainly composed of α-helix, extended strand and random coil compared with cattle, its content is difference; The protein encoded by yak AQP4 or AQP9 contains six transmembrane region. The a-helix in the C-terminal cytoplasmic tail of yak were more than those of cattle; Both AQP4 and AQP9 of yak and of cattle contain the same transmembrane region; Both AQP9 of yak and of cattle contain 5 different phosphorylation sites. There are 2 phosphorylation sites at Ser. One phosphorylation sites is at Thr and another one is at Tyr; Hydrophobicity analysis of AQP4 show that hydrophobicity of 82th amino acid residues in yak is lower than that of cattle; Hydrophobicity analysis of AQP9 show that the hydrophobicity of transmembrane region is higher than others. The ends of the intracellular part show good hydrophily. And the hydrophobicity of 9th and 273th amino acid residues is higher than that of cattle.(4) Both AQP4 and AQP9 mRNA in expressed in the different functional area of yak (cortex, hippocampal, cerebellum and medulla), but the expression level of different species is different. The mRNA expressions of the genes in the yak brain areas were highly significantly lower than those in control cattle (P< 0.001).(5) Immunohistochemical staining of AQP9 in the different functional area (cortex, hippocampal, cerebellum and medulla) of the yak brain is similar to that of cattle; Statistic alanalysis show that both the immunostainingarea (S) and the integral optical densjty (IOD) value of different functional area:Smedulla> Scortex> Scerebellun> Shippocampal and IODmedulla> IODcortex> IODcerebellun> IODhippocampal.both the immunostainingarea (S) and the integral optical densjty (IOD) value of different functional area in yak brain are signifieantly lower than that in eattle brain (P<0.01).(6) Analysis of gene regulatory network shows that AQPs involve in regulation of hypoxia ischemic brain edema as well as many signal transduction pathways, such as HIF-1, TNF, MAPK, VSMC, NF-kβ, CAMs and cGMP-PKG; Among 16 essential genes, differential expression analysis show that 10 of essential genes of yak are significant expression compared with cattle. They are ET-1, HIF-1α, NFAT5, NOS, MMP9, AQP4, AQP9, GST, p38 and VEGF. [Conclusion](1) The coding region sequence of yak AQP4 contains a complete ORF (966 bp) encoding 323 amino acid (gene ID:KM609432); The initiation codon of AQP9 is ATG and its termination codon is TAA. The coding region sequence of yak AQP9 contains a complete ORF (885 bp) encoding 295 amino acid (gene ID:KM820838). Analysis of bio informatics features of yak AQP4 and AQP9 show they have high evolutionary conservation in yak compared with cattle. Lived in the special habitats of Qinghai-Tibet Plateau, the two genes mutated in the primary sturcture (amino acid sequences), second structure (hydrophobicity) and tertiary structure (the distribution of alpha helix in the cytoplasmic tail). The special features of biological information make the "key channel protein" have strong sensitivity to oxygen and other functions, and it plays an important role in the resistance to HACE. Moreover, they provide adequate substrates for energy metabolism of yak on the Qinghai-Tibet Plateau.(2) Low level expression of yak AQP4 and AQP9 reduced the transportation of water and plays an important role in the resistance to HACE.(3) Analysis of gene regulatory network shows that AQPs as well as many signal transduction pathways, such as HIF-1, TNF, MAPK, VSMC, NF-kp, CAMs and cGMP-PKG; Among 16 essential genes, differential expression analysis show that of yak are significant expression compared with cattle. They are ET-1, HIF-1α, NFAT5, NOS, MMP9, AQP4, AQP9, GST, p38 and VEGF.10 essential genes (ET-1, HIF-1α, NFAT5, NOS, MMP9, AQP4, AQP9, GST, p38 and VEGF) of yak may involve in regulation of hypoxia ischemic brain edema on the Qinghai Tibet Plateau under extreme hypoxia stress, but the role of VEGF and GST are to be determined. The specific regulation mechanism need to be further studied.