Association Of Polymorphisms In CRYAA Promoter And CRYAA Protein Interaction Network With Age-Related Cataract

PrefaceCataract is the leading cause of blindness in the world and the main treatment of cataract is surgery. However, the effective surgery is costly, which adds more stress to the individual and the society. New insight into pathogenic mechanisms of cataract will shed light onto its early diagnosis and intervention of high-risk population, which will play an important role in the early prophylaxis.Alpha A crystallin (CRYAA) is one of the major lens proteins, which play central roles in maintaining lens transparency. As a molecular chaperone, CRYAA can prevent protein aggregation in the lens, and protect cells from stress-induced apoptosis.For understanding the function of CRYAA played in delaying the formation of cataract, the following research have been focused on:(I) at the DNA level: association of single nucleotide polymorphisms (SNP) in CRYAA promoter with susceptibility to age-related cataract (ARC);(2) at the protein level:the role of CRYAA protein interaction network played in development of ARC.Patr Ⅰ Association of single nucleotide polymorphisms in CRYAA promoter with susceptibility to age-related cataractPurpose There are differences of CRYAA expression in the lens between ARC patients and the controls. The promoter region sequences modulate gene expression. Hence, the aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the promoter of CRYAA contribute to the susceptibility to ARC.Methods (1) associations of SNPs in the promoter of CRYAA and the susceptibility of ARC:243 ARC patients and 1348 controls were recruited in this study. Five milliliters of peripheral venous blood from all subjects were collected. DNA was isolated from whole blood cells. SNPs in the promoter region (-1-1000) of CRYAA were identified by using the polymerase chain reaction and DNA sequencing. The SHEsis analysis platform was used to calculate the allele and genotype frequencies, the linkage disequilibrium index, and infer haplotype construction; (2) analysis the influence of SNPs on gene expression:the DNA sequence in the promoter of CRYAA (-1-1000) was synthesized and subsequently was cloned into the pGL3-Basic vector; according to the results of SNPs indentified, site-directed mutagenesis was used to get the mutant pGL3-Basic vector at rs7278468. The recombined plasmid were transfected 293T cells, and promoter function analysis were evaluated.Results Six polymorphisms were identified in the promoter of CRYAA: rs3761381, rs3761382, rs79545821, rs13053109, rs7278468, and rs117396767. Only rs7278468 SNP showed a statistically significant relationship with ARC in the analysis of allele frequencies of the patients and control subjects (P<0.05), the minor allele frequency in control group was significantly higher than that in ARC group. All SNPs did not show statistically associated with ARC in the analysis of genotype frequencies (P>0.05). The haplotype analyses demonstrated that the haplotype C-C-G-G-T-C appeared to be a risk factor for ARC (P=0.027, OR=1.258), while the haplotype T-C-A-G-G-C seemed to be protective (P=0.038, OR=0.736). Dual-luciferase report gene assay revealed that the SNP at rs7278468 enhanced the expression efficacy of CRYAA promoter.Conclusions The data indicated that CRYAA polymorphism in promoter is a genetic factor associated with inter-individual differences in ARC risk. Single SNP may have influence on the CRYAA gene expression, while interactions between SNPs could have an additive effect on the risk of ARC: the haplotype C-C-G-G-T-C appeared to be a risk factor, while the haplotype T-C-A-G-G-C seemed to be protective.Part Ⅱ The role of CRYAA protein interaction network played in the development of age-related cataractPurpose The essence of the molecular chaperone is the protein-protein interactions. Hence, the aim of this study is to identify proteins that interacted with CRYAA and to investigate the potential role that protein interactions network played in the formation of cataract using a human proteome (HuProt) microarray.Methods (1) Identification of proteins that interacted with CRYAA:a HuProt microarray composed of 17,225 human full-length proteins with N-terminal GST tags was used to identify protein-protein interactions. The active full-length CRYAA protein corresponding to amino acids 1-173 of CRYAA was recombined. Microarray was incubated with 4ng/μl recombinant full-length CRYAA for 1.5 h (PBST were used as control in control group). After washing three times with PBST, mouse anti-CRYAA monoclonal antibody (1:1000) and goat anti-mouse IgG-Cy3 conjugated antibody (1:200) were added to the microarray slide and incubated for 1 h. The microarray was scanned with an Axon GenePix 4000B Microarray Scanner (Molecular Devices, LLC, Sunnyvale, CA, USA). According to the formular SNR=MeanF532-MeanB532/SDB532, the probes were considered detectable when the signal to noise ratio (SNR) was over 1.2. The identified proteins were subjected to subsequent bioinformatics analysis using the Gene Ontology and DAVID database and proteins interaction network analysis using the string and KEGG database. (2) CRYAA interacted proteins were investigated in 293T cells:the coding DNA sequence of CRYAA was synthesized and subsequently was cloned into the p3xFlag-CMV-7.1 vector. The recombined plasmids were transfected 293T cells, so that Flag-CRYAA complexes protein can be detected. The immunoprecipitated Flag-CRYAA complexes were eluted by anti-Flag M2 beads, and CRYAA interacted proteins were identified by Western blot.Results The HuProt microarray results showed that the signals of 343 proteins were higher in the recombinant CRYAA group than in the control group. The SNR of 127 proteins was≥1.2. The SNR of the following 8 proteins was>3.0:hematopoietic cell-specific Lyn substrate 1 (HCLS1), Kelch domain-containing 6 (KLHDC6), sarcoglycan delta (SGCD), KIAA1706 protein (KIAA1706), RNA guanylyltransferase and 5’-phosphatase (RNGTT), chromosome 10 open reading frame 57 (C10orf57), chromosome 9 open reading frame 52 (C9orf52), and plasminogen activator, urokinase receptor (PLAUR). The bioinformatics analysis revealed 127 genes associated with phosphoprotein, alternative splicing, DNA binding, cell cycle, cytoskeleton, mitochondrion, apoptosis, proteolysis, cellular response to stress, DNA repair and autophagy. The interaction between HCLS1, MAPKBP1, MORG1, HSPB1 and CRYAA were confirmed in 293T cells.Conclusions The CRYAA interacted proteins involved in many biological function:CRYAA maintain physiological state of the membrane by interacting with cytoskeleton; CRYAA is important for lens epithelial growth and viability by interacting with cell cycle proteins; CRYAA protect cell from oxidative damage by interating with cellular response to stress proteins; CRYAA inhibit the apoptosis by interacting with different pathways (MAPK、PI3K/AKt) and DNA repair proteins; CRYAA can control protein degradation by interacting with proteolysis and autophagy proteins.