Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMore than 40 different mutations, in the TBFBIp gene lead to varioustypes of protein aggregates in the cornea including both amyloiddeposits as well as non-amyloid deposits. Here we investigate theprotein composition of two related lattice corneal dystrophy casesand investigate the role of the serine protease HtrA1 in thepathogenesis of this disease.Methods: The amyloid corneal deposits from the two lattice cornealdystrophy cases were isolated from paraffin embedded tissues bylaser capture microdissection. The collected material was treated withtrypsin and analyzed by tandem mass spectrometry. Data weresearched against the Swissprot database using the Mascot algorithmand processed using MS Data Miner software. Spectral counting ofTGFBIp peptides was performed to expose any viabilities in in vivoTGFBIp processing. Furthermore, different genotypes of recombinantexpressed TGFBIp was subjected to proteolysis by the serineprotease HtrA1 and analyzed with SDS-PAGE and MS.Results: Using Exponentially Modified Protein Abundance Index themost abundant proteins in the corneal deposits were compared tohealthy corneal tissue. The amyloid deposits revealed accumulationof serum amyloid p component, clusterin, apolipoproteins A-IV andE and HtrA1. Further, spectral counting of TGFBIp peptidessuggested an increased in vivo processing of TGFBIp associated withthe amyloid deposits compared to healthy tissue. In vitro HtrA1proteolysis of different TGFBIp genotypes showed preference foramyloid forming genotypes as seen in lattice corneal dystrophy.Conclusions: The newly obtained insight into the plaque proteomeassociated with corneal dystrophies has provided new knowledge thatmay help illuminate the mechanism leading to these diseases.Commercial Relationships: Ebbe Toftgaard Poulsen, None;Kasper Runager, None; Michael W. Risør, None; Ida B.Thøgersen, None; Thomas Dyrlund, None; Line R. Thomsen,None; Gordon K. Klintworth, None; Jan J. Enghild, NoneSupport: NH Grant (EY012712) and The Danish National ResearchFoundationProgram Number: 4736 Poster Board Number: C0119Presentation Time: 11:00 AM - 12:45 PMBenzalkonium chloride accelerates amyloid fibril formation incorneal dystrophies in vitroYuichi Kaji 1, 2 , Hisashi Yagi 2 , Yusuke Kato 2 , Yuji Goto 2 , TetsuroOshika 1 . 1 Ophthalmology, University of Tsukuba, Ibaraki, Japan;2 Laboratory of Protein Folding, Institute for Protein Research OsakaUniversity, Osaka, Japan.Purpose: Corneal dystrophies are genetic disorders resulting inprogressive corneal clouding due to amyloid fibril formation derivedfrom the transforming growth factor β-induced (TGFBI) gene.Amyloid fibril formation is influenced by the presence of solventsand surfactants such as sodium dodecyl sulfate (SDS). In the presentstudy, we aimed to reveal the role of benzalkonium chloride (BAC)and SDS in amyloid fibril formation of TGFBI-derived peptides invitro.Methods: Various concentrations of BAC or SDS were added tosolutions of synthetic peptides corresponding to wild-type, Avellinocorneal dystrophy, and lattice corneal dystrophy. The time course ofthe amount of formed amyloid fibrils in the solution wasquantitatively measured using thioflavin T. In addition, the seedingeffect of amyloid fibril formation was evaluated in the presence ofBAC and SDS.Results: For all synthetic peptides, BAC and SDS acceleratedamyloid fibril formation in both de novo and seeding models at 0.01to 0.5 mM and 0.1 to 1.5 mM, respectively. BAC accelerated amyloidfibril formation in the in vitro models of corneal dystrophies.Conclusions: The result indicates that most of the eye dropscontaining BAC may deteriorate corneal dystrophies. Eye drops thatdo not contain BAC would be preferred for patients with cornealdystrophiesCommercial Relationships: Yuichi Kaji, None; Hisashi Yagi,None; Yusuke Kato, None; Yuji Goto, None; Tetsuro Oshika,NoneSupport: This work was supported by the Ministry of Education,Science, Sports, and Culture, Grant for Scientific Research,24370067 (2012-2015), and 24592618 (2012-2015), Japan.Program Number: 4737 Poster Board Number: C0120Presentation Time: 11:00 AM - 12:45 PMExploring the mechanism underlying the protein aggregation instromal corneal dystrophies caused by amyloidogenic and nonamyloidogenicmutants of TGFBIpElavazhagan Murugan 1 , Rajamani Lakshminarayanan 1 , Roger W.Beuerman 1, 2 , Shyam S. Chaurasia 1 , Jodhbir S. Mehta 1, 2 . 1 Tissueengineering and stem cell research group, Singapore Eye ResearchInstitute, Singapore, Singapore; 2 Singapore National Eye Centre,Singapore National Eye Centre, Singapore, Singapore.Purpose: Corneal dystrophies (CD) are a group of inherited disorderscaused by the deposition of proteins in various layers of the cornea.Most of the CDs in the stromal layer of the cornea have beenattributed to the mutations in the transforming growth factor induced(TGFBI) gene with a high propensity in the 4thFAS1 (fasciclin-like)domain of the protein (TGFBIp). Though these mutants exhibitdistinct clinical phenotypes as as fibrillar, non-fibrillar and combinedforms, little or no information is available on their structural andfunctional differences. To understand the mechanism underlying thepathology, we chose representatives from an amyloid (H572R) and anon-amyloid (R555W) phenotype, expressed and purified the 4thFAS1 domains of these proteins and examined them alongwith thewild-type (WT) TGFBIp under various biophysical and biochemicalconditions.Methods: The 4th FAS1 domains of the mutants and the WTTGFBIp were expressed and purified. The ability of the mutants toform oligomers was examined at various conditions of pH andtemperature using circular dichroism spectroscopy. The structuralstabilities of the mutants were analyzed using Urea denaturationstudies. The morphologies of the oligomers formed by heating themutants were studied using electron microscopy.Results: While the WT TGFBIp did not show any conversion insecondary structure, the mutant proteins R555W and H572Rexhibited a clear pH dependent irreversible conversion to oligomersin acidic conditions when heated. While there is a clear conversion atpH 5.5, there is no conversion at pH 7.0. The oligomers formed at pH5.5 were also stable when they were resuspended in pH 7.0. Thestability of the WT and the mutants were tested using Ureadenaturation studies at various pH conditions. Examination usingelectron microscopy also showed that the oligomers of theamyloidogenic H572R were bigger in size with differentmorphologies compared to the non-amyloidogenic R555Woligomers.Conclusions: The mutants clearly show that there is a pH dependentconversion in their secondary structures which leads to their differentmodes of aggregation and hence their distinct pathologies. Theoligomers of the amyloidogenic and non-amyloidogenic mutantsshow differences in their morphologies.Commercial Relationships: Elavazhagan Murugan, None;Rajamani Lakshminarayanan, None; Roger W. Beuerman,Allergan (F), SERI (P), Santen (R); Shyam S. Chaurasia, None;Jodhbir S. Mehta, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.