| Protein is one of the important nutrients for human with a variety of biological activities,such as antibacterial,antioxidant,promoting bone development and enhancing immunity.And chicken eggs are rich in proteins,cheap and easy to obtain.In recent years,the proteomics of different egg parts have been described in detail,and the comparative proteome under various storage conditions and incubation periods have been explored.However,the effects of different environments on egg protein profiles are still unclear.To understand the unique protein patterns from distinct altitudes,in this study,chicken eggs from two altitudes(Tibetan chicken eggs representing high-altitude indigenous breeds and worldwide raised White Leghorn chicken eggs representing low-altitude chicken eggs)were selected to explore the egg white and egg yolk proteomes and their biological functions,and verify the differential antioxidant activity.Then PIT54 was selected as the candidate antioxidant protein through bioinformatics and related study analysis.A series of studies about these two homologous protein were carried out on DNA sequence,protein isolation,mass spectrometry analysis,antioxidant activity,angiogenesis and its mechanism and single nucleotide polymorphism sites in DNA in two altitude chicken eggs.The main findings were as follows:(1)A quantitative proteomics was performed to obtain Tibetan chicken egg white proteome and compared with that of lowland chicken to explore the differential bioactivities.A total of 176 proteins were identified,14 of which showed significantly differential abundances.These differential proteins were found to be related to various health bioactivities,mainly including immune defense,growth and development and antioxidant activity.Among them,the interspecies difference in antioxidant activities was verified by in vitro and in Caco-2 cells,and it was found that both showed antioxidant activities,but lowland chicken egg white protein was much stronger.Prostaglandin-H2 D-isomerase precursor and glutathione peroxidase were considered to be candidate proteins related to antioxidant activity.These findings not only help to explore the impacts of cross-species alteration of reducing oxidative stress damages,but also help us understand that egg white proteins contribute to improving physical activity levels and health outcomes.(2)A comparative study of Tibetan and lowland chicken egg yolk proteins was performed by label-free quantitative proteomics to identify differential egg yolk proteins.A total of 135 proteins were identified and expressions of 19 of these proteins were significantly different between these two groups.The differential proteins were mainly associated with oxidative stress,immunity,energy metabolism and tissue development by GO and KEGG analysis.To further verify the differences in the antioxidant capacity,the antioxidant properties of egg yolk proteins were analyzed in vitro and in Caco-2cells.Both the Tibetan and lowland chicken egg yolk proteins showed antioxidant activities,but the former exerted much stronger effect.Glutathione peroxidase 3 was candidate protein related to antioxidant activities.These results provide new insight and evidence for the molecular mechanisms of improving physical activity levels of egg yolk proteins,especially with regard to the protective mechanisms against oxidative stress.(3)A common collection of antioxidant proteins in chicken egg was selected by constructing non-redundant data sets of egg proteins with manual annotation.With the basic principles of antioxidant activity and few studies on it,PIT54 was selected as a candidate antioxidant activity-related protein which was used for functional verification.Furthermore,the structure and function of PIT54 were analyzed in detail by bioinformatics.It was predicted to contain four repeating SRCR domains and six conserved cysteine residues with scavenger receptor activity and be located on the cell membrane.PIT54 was mainly associated with lipid transport,cell activation and oxygenate response,suggesting that it was indeed involved in hypoxic oxidative stress processes.Chicken PIT54 showed some homology to PIT54 from American crocodile,and without homology to its same function proteins in mammals,haptoglobin.(4)Haptoglobin could bind to hemoglobin and remove broken hemoglobin to protect liver.However,its genes,was not coded by most birds,including chicken.In this study,a novel protein(PIT54)with scavenger activity was purified from chicken egg yolk.To isolate it,egg yolk protein was chromatographed on the DEAE anion column(0-0.5 M Na Cl,50 m M Tris-HCl,p H 8.1)and the hemoglobin binding affinity chromatography column(8 M urea,0.05 M Tris-HCl,p H7.4)in sequence.Such method is simple and in line with animal welfare requirements.Both PIT54 had the abilities of binding hemoglobin and antioxidant capacity,but PIT54 from Tibetan chicken was significantly stronger.Then,N-glycosylations and phosphorylations of T-PIT54 and C-PIT54 were analyzed by mass spectrometry,respectively.A total of three N-glycosylation sites(85N,157 N and 485N)were identified.Among them,157 N was only identified in C-PIT54,and the other two sites(85N and 485N)in the SRCR domain was with higher-abundance in C-PIT54(P < 0.05).This indicated that C-PIT54 has stronger antibacterial ability and helps White Leghorn to adapt to the living environment with many bacteria.Phosphosite Ser411 in the N-terminus of the SRCR domain was detected in both proteins,while the phosphorylation degree of T-PIT 54 was significantly higher than that of C-PIT54(P < 0.05),contributing to promoting T-PIT54 antioxidant ability.(5)In order to study the effects of PIT54 derived from chicken eggs of two altitudes on chicken embryo angiogenesis under normoxia and hypoxia,it is important to simulate a plateau hypoxic environment.On the basis of the commercial incubator,the temperature control system,humidity control system,oxygen control system,carbon dioxide control system and egg turning system were combined to reduce the complex structure and improve the airtightness of the incubator.It was able to accurately and stably simulate the incubation process under different hyoxia environments.The device is simple in operation,low in cost and practical,thus it was used to simulate hypoxic environment for subsequent experiments.Afterwards,the chorioallantoic membranes(CAMs)of the White Leghorn embryo hatched with normoxia and hypoxia,were used as a model to evaluate vascular coverage and the classification of vessel diameter.And D4,D8 and D12 were selected as the time points for subsequent PIT54 treatment.Then,the effects of PBS,T-PIT54 and C-PIT54 on the vascular development of Tibetan chicken and White Leghorn embryos under hypoxic and normoxia were compared and found that both homologous PIT54 showed the greatest effect on chicken embryos on D8.Therefore,NC8,HC8 and HT8 with PBS,C-PIT54 and T-PIT54 treatment,respectively were selected for subsequent mechanisms exploration.(6)PIT54-specific antibodies were prepared using specific fragments of PIT54(CTVNKNLEETETS),and PIT54 was found mainly expressed in the blood and alveoli of chicken embryos,and expressed in the heart,brain and blood vessels with a small amounts,but not in the ligament and liver through western blot using the prepared antibodies.The expression levels and phosphorylation level of VEGFR2 and its upstream and downstream proteins in each group after T-PIT54 and C-PIT54 treatment were detected by western blot,respectively.In hypoxic conditions,C-PIT54 and T-PIT54 could stimulate angiogenesis by significantly increasing the expression of HIF-1?;improves endothelial cell permeability and facilitates budding and migration by enhancing the molecular mechanism of VEGFR2/Src signaling pathway,promote cell proliferation by influencing the molecular mechanism of PI3K/Akt signaling and promoting the phosphorylation of ERK;inhibit apoptosis by increasing phosphorylation of individual p38 proteins.However,these two homologous PIT54 showed interspecific difference on promoting angiogenesis in hypoxic hatched chicken embryos.Both of them have the opposite effect in normoxia hatched White Loghorn chicken embryo to avoid the excessive proliferation of vascular endothelial cells.(7)To explore the molecular mechanism for such bio-activity differences between C-PIT54 and T-PIT54,their DNAs was sequenced and the amino acid sequences and structural differences were predicted by bioinformatics analysis.It was found that the full length of the PIT54 protein gene was 5,186 bp,including 8 exons and 7 introns.The alignment results showed that the introns of T-PIT54 and C-PIT54 dramatically differed,but the CDS sequences of them were highly conserved(99% identify),indicating that PIT54 from Tibetan chicken and white leghorns had genetic stability.Compared with C-PIT54,there were 5 mutations and 5 inserts in the DNA coding sequence of T-PIT54,which should affect the signal peptide sequence,resulting in various transport speed and localization.The basic physical and chemical properties and structural domains were found basically same.However,there were significantly differences in the secondary structures and binding sites between C-PIT54 and T-PIT54.The ?-helix and curl of T-PIT54 increased and the structure become more compact.There were 12 binding sites in both molecules,but some sites have interspecies specificity.For example,amino acid residues at positions 185,186 and 354 were unique binding sites in C-PIT54,and amino acid residues at positions 20,124 and 134 were only in T-PIT54.These differential sites corresponded to distinct parts of the SRCR domain,indicating various molecular structure and biological functions of T-PIT54 and C-PIT54.Such differences might be one of the reasons why their antioxidant activity and angiogenesis activity were different. |
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