| Background: Pathologic myopia has been considered as one of the most common causes of blindness with dramatic increasing prevalence rates.Myopia is influenced by both environmental and hereditary factors.However,the exact pathogenic mechanisms of myopia remain unclear and without effective therapies.And the development of proteomics is essential for exploring pathogenesis,diagnosis,treatments and prognosis of diseases.Our previous studies have found that transthyretin levels serum and vitreous of PM patients significant higher than controls,but the mechanism is still not explained.The two major functions of TTR are transport of T4 hormone produced by thyroid and the transport of retinol through interaction with retinol binding protein 4(RBP4).Furthermore,TTR is a low abundance protein in serum and vitreous,the attempts to purification and identification of TTR is an important foundation for investigating the relationship between PM and TTR.Objective:(1)To further observe the expression levels of TTR in PM patients,also investigate the expression of RBP4 and discuss possible reasons.(2)To explore and optimize the purification for TTR with the application biomimetic affinity chromatography.(3)To make a tentative research of the post-translational modifications characteristics for TTR in PM patients.Method:(1)Collected serum and vitreous of PM patients(38 cases)and emmetropic patients.Concentrations of TTR and RBP4 in the serum,vitreous samples of patients were determined using enzyme linked immunosorbent assay(ELISA)and compared the different levels between two groups.We also explored the differential expressions between subgroups,such as gender,fundus disease,axial lengths and so on.At last,we detected and analyzed the concentrations of biochemical contents in serum between PM patients and controls.We used Graph Pad Prism 6.0software system and the statistic software program SPSS 20.0 for statistical analysis.(2)Biomimetic affinity chromatography(AT23)and a combination of other separated and purified methods for proteins to purify TTR.We describe two strategies for TTR in this work: 1)AT23 was combined with T4 affinity chromatography for purifying TTR.2)We purified TTR by using 40% saturated ammonium sulfate fractionation,followed by AT23 chromatography and ion-exchange chromatography on DEAE Sepharose,eluted by different salt-density gradient.Tricine-SDS-PAGE(18%acrylamide)and WB were performed to analyze the protein purification.(3)Use the Nano LC-ESI MS/MS to detect the PTMs characteristics of TTR in serum and vitreous after separating TTR from samples.Results:(1)We confirmed the up-regulation of TTR in PM patients’ serum and vitreous by ELISA(P<0.05).And the level of RBP4 in PM group was significant higher than control in vitreous,but was not in serum.The ratio of TTR/RBP4 was significant down in serum of PM group(P<0.05).There were three serum biochemical indexes with a significant level(P<0.05).Apo A and GLU in PM patients were lower than controls;to the contrary,ALB was higher in PM group.(2)By exploring the purification process of TTR,we found that the combination of AT23 with T4 affinity chromatography for purifying TTR didn’t work.And the second method exhibited the better purification effect.Combined salting out to AT23 chromatography could remove other proteins including ALB,Ig G and so on,and we obtained fractions containing the TTR were eluted by 150 m M salt-density after submitting to ion exchange chromatography on DEAE.The final purity of TTR was rise from 1% to nearly 30%.(3)Analysis of the potential PTMs of PM and control group revealed that the modified amino acids were different in serum and vitreous,even was from the same patient.The TTR modifications in vitreous were concentrated in amino acids from No.55 to No.68,but in serum,the modified positions were sporadic between No.21 to No.147 amino acids.On the whole,30 and 21 modifications of TTR were identified in serum and vitreous,respectively.The number of modifications identified in vitreous was more than the number of those identified in serum of PM patients,to the contrary,the number of modifications in vitreous was less.We further explored the modifications difference in vitreous between PM patients and controls;found that the number of former was higher than the later.The modification types included Oxidation、Dioxidation、Kynurenin 、 Formylation 、 Propionylation 、 Guanidinylation 、 Glycine addition were in higher frequency than those contained Acetylation、Nitro、Octanoylation、sulfofication and so on.Conclusion:(1)The concentrations in serum and the vitreous of PM patients were significantly higher than in emmetropic patients,we suggested that TTR is a potential marker and possible pathogenic factor in PM.The level of TTR and Axial lengths was in positive correlation.(2)The lower TTR/RBP4 ratio and GLU level in PM serum might be a clue to explore the relationship between DM and PM.(3)Combined salting out to biomimetic affinity chromatography,followed by anion exchange chromatography is an effective purification for TTR;meanwhile we are looking for the purification that may be even more optimal.(4)We speculate that the different modifications between serum and vitreous might be related to the microenvironment of them.Furthermore,preliminary exploring the characteristics of TTR in serum and vitreous of PM that laid a foundation for further researches on metabolisms and pathogenesis for PM. |
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