The Proteome Study Of Congenital Inherited Cataract

Congenital cataract is the most frequent cause of children blindness. In developed countries, the prevalence of congenital cataract ranges from 1 to 4 per 100,000 children; in the developing world, it ranges from 5 to 15 per 100,000. It is estimated that 130,000 to 200,000 children are blind from bilateral congenital cataract in the world. About 20,000 to 40,000 new cases of bilateral developmental cataracts are added each year.Genetic factors play the major role of congenital cataract. So far, 12 loci and 15 specific genes with congenital isolated cataract have been intentified at least, including three kinds of crystallins, membrane proteins, cytoskeletal proteins, transcriptor factors, and unknown function protein such as Lim2. However the actor of gene function was protein indeed, all of the phenotypes were not interpreted completely from the gene level. And the molecular biological research fields entered into the proteome era. Lens proteomics of congenital cataract are fashionable in the world.Animal model was a powerful tool in the proteomics research fields because of the comparative results and controllable experimental condition. A recessive mice model was adopted. The histology and morphology of cataract were investigated. The mice were validated with a spontaneous Crygs mutation. However there is no study about the changes of downstream proteins induced by mutated gene. The aim is to search different proteins between cataract and normal lens by novel proteomics techniques. It is to elucidate the relationship between mutated gene and proteins in the development of congenital cataract. We found a cataract family inherited as X-linked dominant traits. We also analyzed the serum proteome of cataract and healthy persons to search markers of cataract for diagnosis.Part I : The change of histology and ultra structure in congenital inherited cataractObjectiveTo analyze the histology and ultra structure of crystalline lens in congenitalCrygs mutated mouse. MethodsWe applied a recessive mice model with spontaneous Crygs mutation in the KUNMING outbred mice. Mutant mice and normal mice aged 5 weeks were respectively as experimental and control group. All mice were underwent ocular observation and slit lamp microscope photography after pupil dilation. Then we killed the mice by vertebra-dislocation method and extracted the eyeballs. The eyeballs were fixed. Paraffin-embedded sections were stained with hematoxylin and eosin, and then were observed by light microscope. The lenses were extracted for transmission and scanning electron microscopy. ResultsCompared to normal group, Crygs mutated mice had symmetric nuclear cataract of both eyes. The lens nucleus was completely covered by white opacities. However, the periphery was transparent. Histological examination of lens showed Morgagnian bodies in mutant mice. Superficial cortex fiber aligned loosely and had vacuole-like degeneration. Epithelial cells in the equatorial region seemed to over-proliferate and immigrate anteriorly and posteriorly. Nucleus of lens was pink deeply stained, with the shorter fiber losing their normal arrangement. Big vacuole was seen underneath the posterior lens capsule. By transmission electronic microscopy, mutant mice had enlarged intercellular spaces, liquefied cytoplasm with the swollen mitochondria. By scanning electronic microscopy, the irregular arrangement of cortex and nuclear fibers and the abnormal ball-socket junctions were observed, with Morgagnian bodies and vacuole-like degeneration. ConclusionsMutation in Crygs gene can accelerate the proliferation and degeneration of lens epithelium. It also disturbs cell conjunctions and the arrangement of lens fiber, then leading to the formation of cataract. γS crystallin locates in the cortex region. However, Crygs mutation leads to nuclear cataract. It suggests that nuclear changes maybe due to other proteins aroused by γS crystallin. We should undergo further proteins analysis. Part II: Comparative analysis of the proteome of lens in congenital inherited cataract miceObjectiveTo separate total lens proteins of congenital inherited cataract in mice and observe the alteration of proteins after Crygs gene mutation. MethodsA spontaneous Crygs mutated mice transmitted as a recessive trait were studied. We analyzed differential displayed proteomics of cataract and normal crystalline lens of 5 weeks' mice. The proteins were separated using immobilized pH gradient (IPG) two-dimensional electrophoresis (2-DE) and colloidal Coomassie Brilliant Blue (CBB) staining. Image analysis was carried out using PDQuest 7.30 software package. Partial significant differentially proteins in gel were identified by matrix assisted laser adsorption/ionization-time of flight-tandem mass spectrometry(MALDI-TOF-MS/MS). ResultsDifferent amount of sample can separate proteins of different abundance. It could separate the low-abundance proteins when we applied a higher sample and the high-abundance proteins, a lower sample. We performed 2-DE three times to observe the reproducibility. One gel acted as the reference gel. When a 882μg sample was added, the mean matched ratio was 78.7% and 81.3% and we had detected 417 ± 53 spots and 370 ±41 spots in cataract and normal lens respectively. As a 190μg sample, the mean matched ratio was 90.4% and 92.5% and we had detected 60 ± 7 spots and 57 ± 5 spots in cataract and normal lens respectively. When normal gel acted as the reference gel, the mean matched ratio was 67% versus 53% (882μg) and 93% versus 89% (190μg) in normal and cataract lens. The average positional deviation of the matched spots among normal and cataract profiles was (0.92 ± 0.15) mm in IEF direction and (1.28 ± 0.21) mm in SDS-PAGE direction. 16 chosen spots were identified by MALDI-TOF/TOF, where included BFSP/filensin, γ S, γ F, βA1, β B1, β B2, α B and so on. In crystalline lens of mutant mice, γS and beaded-filament structure protein (BFSP/filensin) were not detected. γF was down expressed (<4 fold) while β A1, β B1, β B2 and αB were over expressed (>4 fold) in mutant cataract. The latter proteins have less MW than normal. It suggested they were perhaps truncated. ConclusionsThe protein profiles of congenital inherited cataract displayed obvious differences compared to that of normal lens. Lower sample can well separate thehigh-abundance proteins of crystalline lens. Mutant gene can alter the change of the downstream proteins. 2-DE and mass spectrometry can help to assess and analyze the function of proteins as a novel approach. The mutant mice is a good model for the research of γ S. The mutated Crygs gene can lead to the abnormity of γ S crystallin, which can influence other proteins such as skeletonal protein (BFSP/filensin) and truncated crystallins (γF, βA1, βB1, βB2 and α B) secondarily. The hypothesis is that γ S crystallin can inhibit the activity of endogenous proteases. Just like α B crystallin, γ S crystallin is not only a lens structural protein, but also a functional protein.Part III: Study of serum proteins of a congenital cataract family by comparative proteome analysisObjectiveTo compare serum total proteins of congenital cataract patients with normal human. MethodsA congenital cataract family was analyzed. Family members received with slit lamp examination. We obtained their peripheral blood and collected the serum. Comparative proteomic analysis with 2-DE separation was utilized to compare the total protein expression profiles of serum between normal and patients. The proteins were separated using immobilized PH gradient (IPG), two-dimensional electrophoresis (2-DE) and silver staining. Image analysis was carried out using ImageMaster software package. ResultsWe found a cataract family inherited as X-linked dominant traits. The lens opacity is perinuclear-shaped with a transparent embryonic nuleus. There was no distinct difference in expression profiles of total serum between cataract and normal age-matched groups. ConclusionsA Chinese family with X-linked dominant lamellar cataract was detected. Serum proteins of two groups showed no differences. There were two possible reasons. Oneis that the crystalline lens has no vessels and is separated from blood. The second, low-abundance of serum proteins are difficult to separate. Mutant gene maybe only operated in the tissue of crystalline lens. Proteomics analysis of crystalline lens tissue is preferable to congenital cataract research.