Screening and identification of CRYAA mutationsinARCpatients
Genomic DNA samples were isolated from peripheral bloodsamplesof300 ARC patients and 100 normal subjects. The SSCPassayshowedmobility shift pattern in a 63-year-old male ARCpatient(S220) inexon 2 of CRYAA gene(Fig. 1A). Such changes werenotobservedamong 100 normal controls. Sequencing ofamplifiedproductsrevealed a novel heterozygous G > Anucleotidesubstitution inexon 2 of CRYAA (c.249G > A,NG_009823.1), whichdid not changethe amino acid sequence(Glutamicacid, Glu) at codon83(p.E83E)(Fig. 1B). The Glu83 of CRYAA genearehighlyconserved among 8 species(Fig. 1C).
Protein expressions, distribution andchaperonefunctioninfluenced by CRYAA-E83E
We construct CRYAA-WT and CRYAA-E83E plasmids toinvestigatethefunction of the novel silent mutation of CRYAA-E83E.In HLEB-3cells, compared to blank vector, transfection ofCRYAA-WTandCRYAA-E83E showed significant increasedCRYAAgene(Fig. 2A)andprotein(Fig. 2B) expression levels by RT-PCRandIB,respectively; compared to CRYAA-WT, CRYAA-E83Eshowedhigherexpression levels of CRYAA gene and protein levelsdetectedbyRT-PCR (Fig. 2A)andIB(Fig. 2B), respectively. Toinvestigatetheprotein distribution of CRYAA, protein expressionlevelsinfluencedby CRYAA-E83E in nuclear and cytoplasmic proteinsof HLEB-3 cellswere analyzed. Histone H3 was used as internalreferenceof nuclearproteins. β-actin was used as internal referenceofcytoplasmicproteins. Compared to blank vector, theexpressionlevels of CRYAAcontributed by CRYAA-WT and CRYAA-E83Eweresignificantly higher innucleus and cytoplasm. Compared toCRYAA-WT,the protein expressionlevels of CRYAA by CRYAA-E83E waslower inboth nuclear andcytoplasmiclysates(Fig. 2C).
CRYAA is widely regarded as the small heat shockproteinwhichmainly suppress the aggregation of differentclientproteins.CRYAA-WT and CRYAA-E83E plasmids were transfectedinto HLEB-3cells. Cell lysates were centrifuged into solubleandinsolublefractions, the insoluble fraction indicates theexpressionlevelsof aggregated client proteins. Aggregation ofGAPDH, α-SMA,FN,laminin γ2 proteins in HLE B-3 cell lysates weredetected byIB.Compared to blank vector, HLE B-3 cells transfectedwithCRYAA-WTshowed higher expression levels of total, solubleandinsolubleCRYAA detected by IB; compared to CRYAA-WT, HLEB-3cellstransfected with CRYAA-E83E showed higher solubleCRYAAexpressionlevels and similar insoluble CRYAAexpressionlevels(Fig. 2D). Compared to blank vector,HLEB-3cells transfected with CRYAA-WT showed similar total,solubleandinsoluble GAPDH expression levels; compared to CRYAA-WT,HLEB-3cells transfected with CRYAA-E83E showed similartotalGAPDHexpression levels, lower soluble GAPDH expressionlevelsandsimilar insoluble GAPDHexpressionlevels(Fig. 2D). Compared to blank vector,HLEB-3cells transfected with CRYAA-WT showed similar total,solubleandinsoluble α-SMA expression levels; compared to CRYAA-WT,HLEB-3cells transfected with CRYAA-E83E showed similartotalα-SMAexpression levels, lower soluble and insolubleα-SMAexpressionlevels (Fig. 2D). Compared to blank vector,HLEB-3cells transfected with CRYAA-WT showed higher totalFNexpressionlevels, lower soluble FN expression levels andhigherinsoluble FNexpression levels; compared to CRYAA-WT, HLE B-3cellstransfectedwith CRYAA-E83E showed lower total FNexpressionlevels, highersoluble FN expression levels and lowerinsoluble FNexpressionlevels (Fig. 2D). Compared to blank vector,HLEB-3cells transfected with CRYAA-WT showed similar total,solubleandinsoluble laminin γ2 expression levels; comparedtoCRYAA-WT,CRYAA-E83E had similar total, soluble andinsolublelaminin γ2expression levels(Fig. 2D).
Morphology and chaperone function influencedbyCRYAA-E83Etreated with 42℃
To further investigate the chaperone functioninfluencedbyCRYAA-E83E, HLE B-3 cells transfected with CRYAA-WTandCRYAA-E83Eplasmid were cultured in medium at 37℃ for24 h,then changedinto 42℃ for 4 h, with those cultured at37℃ for4 h asnegative control. After treated, cells shrinked,becamesmall andturned round, and vacuoles were located aroundthecells(Fig. 3A). Aggregation of CRYAAandGAPDHwere detected by IB through chaperone function. Comparedtoblankvector, the expression levels of total, soluble andinsolubleCRYAAwere higher in HLE B-3 cells transfected withCRYAA-WTandCRYAA-E83E; compared to CRYAA-WT, HLE B-3 cellstransfectedwithCRYAA-E83E showed lower soluble CRYAA expressionlevels andhigherinsoluble CRYAA expressionlevels(Fig. 3B). Compared to blankvector, HLEB-3cells transfected with CRYAA-WT showed similartotal andsolubleGAPDH expression levels, slightly lower insolubleGAPDHexpressionlevels; compared to CRYAA-WT, HLE B-3 cellstransfectedwithCRYAA-E83E showed similar total GAPDH expressionlevels,lowersoluble GAPDH expression levels and higherinsolubleGAPDHexpression levels (Fig. 3B).
Morphology and protein expression in HLE B-3 cellsandhumananterior lens capsules treated by UV
To investigate the influence of UV treatment onmorphologyandprotein expressions of HLE B-3 cells, cells weretreated withUVfor 30 min or 1 h, with cells withouttreatmentasnegative control. After UV treatment, HLE B-3cellsbecameirregular in shape and flatten, particularly thosetreatedwith UVfor 1 h (Fig. 4A). In the earlystage(UV30 min), apoptosis with nuclear chromationcondensationandfragmentation as well as apoptotic bodieswereobserved(Fig. 4A). In the late stage (UV1 h),cellsbecame more flatten, and the apoptotic cells mightbephagocytizedby adjacent cells (Fig. 4A). Compared tocellswithout UVtreatment, HLE B-3 cells treated with UV showedlowerexpressionlevels of CRYAA, FN and α-SMA, and higherexpressionlevels oflaminin γ2 and N-cadherin byIB(Fig. 4B).
We futher collected human ALCs of nuclear grade Ⅲ from20ARCpatients. Human ALCs were treated with UV for 1 handculturedin medium at 37℃ for another 24 h, and thentreatedwith UVagain for 30 min. Human ALCs without treatedwere asnegativecontrol (without). A large number of lens epithelialcellsunderanterior capsules were observed. Cells becameirregular,flattenand vacuolization, the nucleus boundaries wereunclear,which issimilar to the changes of HLE B-3 cells treatedwith UV.Cellapoptosis was induced (Fig. 4C). IB of human ALCs showedlowerexpressionlevels of CRYAA and N-cadherin, and higherexpressionlevels oflaminin γ2 after treated withUV(Fig. 4D).
Morphology and chaperone function influencedbyCRYAA-E83Etreated with UV
To further investigate the chaperone functioninfluencedbyCRYAA-E83E at different conditions, CRYAA-WTandCRYAA-E83Eplasmids were transfected into HLE B-3 cells.Thentreated HLE B-3cells with UV for 30 min, cellswithouttreatment as negativecontrol. HLE B-3 cells without anytreatmentand cells transfectedwith blank vector became irregularand flattenafter treated withUV. A large number of apoptotic bodieswereobserved in HLE B-3cells transfected with CRYAA-WT andCRYAA-E83Eafter UV treatment(Fig. 5A). In HLE B-3 cells treatedwithUVfor 30 min, compared to blank vector, cellstransfectedwithCRYAA-WT and CRYAA-E83E showed significantlyhigherCRYAAexpression levels; compared to CRYAA-WT, the expressionlevelsofCRYAA contributed by CRYAA-E83Ewerehigher(Fig. 5B). Compared toblankvector,Bcl-2/Bax ratio by CRYAA-WT was higher, suggestingCRYAA-WTmighthave function of anti-apoptosis; compared to CRYAA-WT,HLEB-3cells transfected with CRYAA-E83E showed higherBcl-2/Baxratio(Fig. 5B), suggestingCRYAA-E83Emutationmight increase the ability of anti-apoptosis.Aggregationof CRYAAand GAPDH protein in HLE B-3 cell lysates weredetected byIBthrough chaperone function assay. Results showed that,comparedtoblank vector, the expression levels of total CRYAAwerehighercontributed by CRYAA-WT and CRYAA-E83; compared toCRYAA-WT,theexpression levels of total, soluble and insoluble CRYAAwashigherby CRYAA-E83E (Fig. 5C). Compared to blank vector,HLEB-3cells transfected with CRYAA-WT showed similar total,solubleandinsoluble GPADH expression levels; compared to CRYAA-WT,HLEB-3cells transfected with CRYAA-E83E showed lowersolubleGAPDHexpression levels and higher insoluble GAPDHexpressionlevels(Fig. 5C). Suggesting the impairedchaperonefunctionof CRYAA-E83E. To investigate the expressionlevels ofhistone H3,we collected BM of HLE B-3 cells treated withUV for30 min,with BM without treated as negative control. Inthe BMof untreatedHLE B-3 cells, compared to blank vector,theexpression levels ofhistone H3 contributed by CRYAA-WT werelower;compared to CRYAA-WT,the expression levels of histone H3byCRYAA-E83E were similar(Fig. 5D). CBB staining (bottom)ofBMproteins was used to beinternalreference(Fig. 5D). In the HLE B-3 cellBMtreatedwith UV, the expression levels of histone H3 levelbyCRYAA-E83Ewere significantlyup-regulated(Fig. 5D). To investigatetheexpressionlevels of secretory CRYAA, CRYAA-WT andCRYAA-E83Eplasmids weretransfected into HLE B-3 cells. Aftertreated with UVfor30 min, the supernatant of HLE B-3 cells wascollected,withsupernatant without treated as negative control. Inthesupernatantof HLE B-3 cells, compared to blank vector,theexpression levels ofCRYAA were higher contributed by CRYAA-WTandCRYAA-E83E; comparedto CRYAA-WT, the expression levels of CRYAAbyCRYAA-E83E were lowerbefore treated, but higher after treatedwithUV for 30 min(Fig. 5E).