pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

1 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 www.na u e.com/scien i ic epo s pH Induced Con o ma ional T ansi ions in he T ans o ming G ow h Fac o β-Induced P o ein (TGFβIp) Associa ed Co neal Dys ophy Mu an s Ela azhagan Mu ugan1,2, Anandalakshmi Venka aman1, Zhou Lei3, Vic o ia Mou e 1, Rayne Rui Yi Lim1, Nandhakuma Mu uganan ham4, Eunice Goh4, Ga y Swee Lim Peh1,2, Roge W. Beue man2,4,5,6, Shyam S. Chau asia1,2,5, Lakshmina ayanan Rajamani2,4,5 & Jodhbi S. Meh a1,2,5,6 Mos s omal co neal dys ophies a e associa ed wi h agg ega ion and deposi ion o he mu a ed ans o ming g ow h ac o -β induced p o ein (TGFβIp). The 4 h_FAS1 domain o TGFβIp ha bo s ~80% o he mu a ions ha o ms amyloidogenic and non-amyloidogenic agg ega es. To unde s and he mechanism o agg ega ion and he di e ences be ween he amyloidogenic and non-amyloidogenic pheno ypes, we exp essed he 4 h_FAS1 domains o TGFβIp ca ying he mu a ions R555W (non- amyloidogenic) and H572R (amyloidogenic) along wi h he wild- ype (WT). R555W was mo e suscep ible o acidic pH compa ed o H572R and displayed a ying chemical s abili ies wi h dec easing pH. The mal dena u a ion s udies a acidic pH showed ha while WT did no unde go any con o ma ional ansi ion, he mu an s exhibi ed a clea pH-dependen i e e sible con e sion om αβ con o ma ion o β-shee oligome s. The β-oligome s o bo h mu an s we e s able a physiological empe a u e and pH. Elec on mic oscopy and dynamic ligh sca e ing s udies showed ha β-oligome s o H572R we e la ge compa ed o R555W. The β-oligome s o bo h mu an s we e cy o oxic o p ima y human co neal s omal ib oblas (pHCSF) cells. The β-oligome s o bo h mu an s exhibi a ia ions in hei mo phologies, sizes, he mal and chemical s abili ies, agg ega ion pa e ns and cy o oxici ies. Co neal Dys ophies a e inhe i ed p o ein agg ega ion diso de s cha ac e ized by he deposi ion o mis olded p o eins agg ega es in a ious laye s o he co nea1–3. Mos dys ophies in he co neal s omal egion a e asso- cia ed wi h he mu a ions in he ans o ming g ow h ac o β -induced p o ein (TGFβ Ip). TGFβ Ip agg ega ion and deposi ion occu s only in he co nea, hough i is p esen in abundance in a ious connec i e issues4–7. The ma u e TGFβ Ip, a 660aa p o ein has an N- e minal cys eine- ich EMILIN-like (EMI) domain, ou ascicilin-like (FAS1) domains and an in eg in-binding RGD mo i a he C- e minus8. TGFβ Ip-associa ed co neal dys ophies a e pheno ypically he e ogeneous, inhe i ed in an au osomal dominan manne 1,9,10 and a e classi ied as la ice, g anula , combined la ice and g anula , Reis-Buckle and Thiel-Behnke co neal dys ophies1,2,11,12. So a , 64 single amino acid mu a ions associa ed wi h dis inc pheno ypes ha e been epo ed4,13,14. Among he ou FAS1 domains o TGFβ Ip, he 1s and 4 h FAS1 domains ca y he disease ela ed mu a ions, wi h ~80% o he mu a- ions esiding in he 4 h_FAS1 domain11. In la ice co neal dys ophies (LCD), he p o ein agg ega es appea as 1Tissue Enginee ing and S em Cell G oup, Singapo e Eye Resea ch Ins i u e, Singapo e. 2Duke-NUS G adua e Medical School, Singapo e. 3P o eomics and Mic oanalysis labo a o y, Singapo e Eye Resea ch Ins i u e, Singapo e. 4Ocula Chemis y and An i-In ec i es, Singapo e Eye Resea ch Ins i u e, Singapo e. 5Depa men o Oph halmology, Yong Loo Lin School o Medicine, NUS, Singapo e. 6Singapo e Na ional Eye Cen e, Singapo e. Co espondence and eques s o ma e ials should be add essed o J.S.M. (email: [email p o ec ed]) o L.R. (email: [email p o ec ed]) Recei ed: 17 June 2015 Accep ed: 12 Feb ua y 2016 Published: 31 Ma ch 2016 OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 la ice lines o amyloid ib ils (amyloidogenic). In g anula co neal dys ophies (GCD), hey appea as g anula , non-amyloidogenic deposi s11–16. Bo h pheno ypes display signi ican di e ences in mo phology, agg ega ion and inc o ial p ope ies. Howe e , he mechanisms adop ed by hese pheno ypes in o ming highly dis inc ul as- uc u es, emains o be elucida ed. I has been epo ed ha he abili y o o m highly o de ed agg ega es such as amyloids, esides wi hin he polypep ide chains a he han he whole p o ein17. Hence, we chose o explo e he c ucial 4 h_FAS1 domain (135aa) as a ep esen a i e o he ull-leng h TGFβ Ip. Bioin o ma ics analyses o he agg ega ion p opensi ies o a ious egions o TGFβ Ip ha e also shown ha he 4 h_FAS1 domain ha bo s egions o high agg ega ion p opensi ies18,19. Also, homology-based modelling s udies ha e shown ha he 4 h_FAS1 domain displays he p ope ies o he ull-leng h TGFβ Ip 20. In ou s udy, we aim o delinea e he di e ences be ween he amyloidogenic and he non-amyloidogenic mu an s and be ween he mu an s and wild- ype (WT) TGFβ Ip o explain he physiological a ia ions exhibi ed by hese pheno ypes. We had p e iously epo ed he cloning, exp ession and pu i ica ion o he 4 h_FAS1 domain o ou TGFβ Ip mu an s21. Ou s udies indica ed ha unde physiological pH, he mu an s a e mo e s able han he WT. He e, we chose o examine he e ec s o ac o s like empe a u e and pH on he p ope ies o he 4 h_ FAS1 domains o TGFβ Ip ha bo ing mu a ions o he amyloidogenic and non-amyloidogenic pheno ypes. The amyloidogenic H572R (LCDI/IIIA), disco e ed in Thai22 and Chilean popula ions23 wi h ages o onse anging a ound mid- wen ies, is cha ac e ized by cen al sub-epi helial needle-like la ice lines and polymo phic an e io s omal opaci ies. The highly ubiqui ous non-amyloidogenic R555W (GCDI/II)24–26 appea s as od shaped o g anula bodies wi h sha p bo de s ound in he cen al co neal s oma27,28. We aimed o examine he e ec s o hese cha ge modi ying mu a ions on he domains, hei agg ega ion, hei sensi i i y o pH and empe a u e. Agg ega ing p o eins a e ound o be mo e suscep ible o acidic pH and inc ease in empe a u e29,30. E en in TGFβ Ip, i has been shown ha he R124H mu a ion induces localiza ion o TGFβ Ip o lysosomes wi h an acidic en i onmen 31. In he p esen s udy, we ha e in es iga ed he e ec s o acidic pH, dena u an s and empe a u e on he seconda y s uc u e and con o ma ional s abili y o he domains. The cy o oxici ies o he agg ega es we e also s udied in p ima y human co neal s omal ib oblas s (pHCSF). Resul s E ec s o biochemical and biophysical ac o s on WT and mu an s. The na i e 4 h_FAS1 domain (Fig.1a) o TGFβ Ip (Genbank_ID-NM_000358; P o ein_ID:-NP_000349) and he mu an s R555W and H572R we e cloned and pu i ied (Fig.1b) as desc ibed p e iously21. The es ima ed pI alues o he WT, R555W and H572R domains we e 6.53, 6.32 and 6.65 espec i ely. The amino acid subs i u ions (R→ W and H→ R) we e associa ed wi h changes in cha ge and hyd ophobici y as lis ed in he able (Table1). E ec o pH on he seconda y s uc u es o he WT and mu an s. The CD spec a o he WT and he mu an s a pH 7 (Fig.1c–e) showed nega i e minima in he n – π * egion (222 nm) and a weak shoulde a he π –π * egion (207 nm) co esponding o hei mixed α -helical and β -shee con o ma ions. Unde acidic condi ions, he e we e disce nible di e ences be ween he WT and mu an s in hei seconda y s uc u e. The non-amyloidogenic R555W was mo e sensi i e o pH compa ed o he WT and amyloidogenic H572R. The WT (Fig.1c) and H572R (Fig.1e) emained unchanged unde neu al and acidic pH. The R555W mu an displayed an inc ease in he CD in ensi y a 222 nm and 207 nm wi h dec ease in pH (Fig.1d). CD in ensi ies a 222 nm a a ying pH alues (Fig.1 ) showed ha R555W, was mo e sensi i e o pH and he pH- esponse o H572R was simila o he WT p o ein. We also incuba ed he mu an s in acidic pH o 1 week and ollowed hei agg ega ion/ oligome iza ion by ThT luo escence (Supplemen a y Fig. S1). Howe e , no signi ican con e sion was obse ed as seen om he co esponding CD spec a o WT and H572R. A pH 2.75, R555W showed a pa ial con e sion o β -shee . Compa ed o he amyloid ib il pep ide pN622K, almos no inc ease in luo escence was obse ed o WT and R555W. The sligh inc ease in luo escence co esponding o pH 2.75 and pH 3.25 o H572R did no show a co esponding con e sion in he CD spec a. E ec pH on he mal dena u a ion o WT and mu an s. Con o ma ional ansi ion o he domains unde going he mal dena u a ion was examined by hea ing hem om 20 °C o 70 °C, a neu al and acidic pH condi ions. A pH 7 and pH 8, o bo h he mu an s and he WT, no well-de ined ansi ion was obse ed wi h inc easing empe a u es (Fig.2a– ). Fo bo h he mu an s, he ampli ude o he nega i e minima a 222 nm dec eased upon hea ing (Fig.2c– ) and a weak hys e esis was obse ed when cooled. Wi h acidic pH, while he WT showed no appa en changes in he seconda y s uc u e wi h inc easing empe a u e (Fig.3a–c), bo h he mu an s displayed a clea ansi ion om monome ic α /β -s uc u e o β -shee (Fig.3d–i). Single wa eleng h scan a 222 nm indica ed a clea sigmoidal ansi ion o bo h mu an s unde acidic condi ions when compa ed o he WT, which was unpe u bed by he changes in pH and empe a u e (Fig.4a–e). The mal dena u a ion expe - imen s we e also done by hea ing he domains om 20 °C o 90 °C (Supplemen a y Fig. S2). Though p e ious s udies ha e obse ed dena u a ion o WT abo e 60 °C 32, we did no obse e any con o ma ional ansi ions o β -shee e en a e hea ing o 90 °C. The mid-poin o he no malized sigmoidal cu e de ines he ansi ion em- pe a u e (T ) whe ein he con e sion o a monome ic α /β -s uc u e o he β -s uc u ed oligome s was obse ed. The ansi ion o he domains a di e en pH condi ions was simila o he samples hea ed o 70 °C and he T lied be ween 35–58 °C. Hence all he subsequen he mal dena u a ion expe imen s we e pe o med by hea ing he domains up o 70 °C. The non-amyloidogenic R555W showed a ma ked sensi i i y o pH and displayed a highe he mal ins abili y compa ed o he H572R. Though H572R mu an displayed a clea pH-dependen con e sion o β -s uc u e when hea ed, he T was highe han R555W. A di e ence in T o 5–12 °C is obse ed a a ious pH condi ions. Mild p ecipi a ion was obse ed in he samples a e hea ing, which co ela es wi h he change in www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 in ensi ies obse ed in he CD spec a. When he concen a ion o he sample was inc eased ( om 0.6 mg/ml o 1.2 mg/ml~75 μM), he u bidi y and p ecipi a ion inc eases. U ea dena u a ion o non-amyloidogenic R555W. The yp ophan esidue in R555W allowed us o measu e he emission luo escence. Examina ion o he emission luo escence a ~332 nm o R555W in acidic pH showed a signi ican dec ease in emission in ensi y wi h dec easing pH, howe e he emission maxima emained unchanged (Fig.5a). To ob ain a be e insigh in o he e ec o pH on R555W, we moni o ed he u ea-induced un olding o R555W. Inc easing he u ea concen a ion p og essi ely shi s he emission maxima (~332 nm) o longe wa eleng hs (~352 nm), sugges ing a clea ansi ion om olded o un olded con o ma ions (5b–e). To con i m e olding o un olded R555W, he un olded p o ein a di e en pH condi ions (pH 3.0, pH 4.5, pH 5.5 and pH 7.0) was dilu ed app op ia ely and emission spec a we e eco ded. The emission maxima (λ max) plo - ed wi h he un olded and e olded domains we e supe imposable (Supplemen a y Fig. S3). (Fig.5b–e). A clea e e sal in luo escence maxima om ~352 nm o ~332 nm was obse ed he eby allowing us o es ima e he he modynamic s abili y o he mu an p o ein in a ious pH. Figu e5 –i shows u ea dena u a ion cu es plo ed as ‘ ac ion un olded (yU) s inc easing u ea concen a ions’ as moni o ed by he changes in emission maxima Figu e 1. Biochemical and biophysical p ope ies o he na i e 4 h_FAS1 domains o he wild- ype and mu an TGFβIp. (a) Schema ic ep esen a ion o he domain a angemen and bounda ies o he ull-leng h TGFβ Ip and 4 h_FAS1 domains o he wild- ype, non-amyloidogenic (R555W) and amyloidogenic (H572R) mu an s used in he s udy. (b) SDS-PAGE gel showing he pu i ied ac ions o he 4 h_FAS1 domains o he WT, R555W and H572R mu an s. (c–e) Fa UV CD spec a o he 4 h_FAS1 domains o WT (d), R555W (e) and H572R ( ) incuba ed o 16 hou s a acidic pH condi ions (pH 3, 4.5, 5.5 and 7). The R555W mu an displayed clea changes in he CD spec a a 222 nm and 207 nm wi h dec ease in pH ( ). The CD in ensi y a 222 nm dec eased wi h dec ease in pH con i ming he un olding o he seconda y s uc u es. The CD spec a o he WT (c) and H572R (e) mu an emained almos unchanged. Plo ing he in ensi ies a 222 nm a a ying pH (g) showed ha he non-amyloidogenic pheno ype, R555W, was mo e sensi i e o pH and he amyloidogenic pheno ype, H572R, emained mo e s able o pH changes a oom empe a u e. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 (Δλ max) a a ious pH alues (Supplemen a y Fig. S3) o he R555W mu an . The he modynamic pa ame e s de i ed om u ea dena u a ion a e shown in he able (Table2). A signi ican dec ease in ee ene gy o un old- ing om 13.8 ± 0.8 kJ/mole o 7.108 ± 1.5 kJ/mole was obse ed om pH 7.0 o pH 4.5 o he R555W mu an . Howe e , o he H572R mu an , luo escence s udies could no be pe o med because o he absence o a yp- ophan esidue. Cha ac e iza ion o he β-oligome s o amyloidogenic and non-amyloidogenic mu an s. The amyloidogenic and non-amyloidogenic mu an s displayed a clea ansi ion o an all β -shee con o ma ion when hea ed unde acidic condi ions. Con e sion o an all β -shee con o ma ion could indica e β -oligome o ma- ion17. A de ailed in es iga ion o he β -oligome s om wo mu an s was he e o e pe o med o alida e and cha ac e ize he p oposed β -oligome s. Con i ma ion o β-oligome s o med by he mu an s and cha ac e iza ion o he β-oligome s by TEM and DLS. TEM examina ion o he β -oligome s e ealed ha while no pa icles we e isible o he WT (da a no shown), bo h he mu an s displayed pa icles alida ing ou p oposal. The β -oligome s o he H572R and R555W displayed a ying sizes and mo phologies (Fig.6a,b). The β -oligome s o he non-amyloidogenic R555W we e homogeneous, displayed smoo he edges and measu ed ~4–8 nm (mean diame e ~5.1 ± 1.79 nm) (Fig.6a). The β -oligome s o he amyloidogenic H572R we e mo e he e ogeneous, displayed ugged edges and we e la ge , measu ing 10–40 nm (mean diame e ~19.1 ± 4.9 nm) (Fig.6b). The β -oligome s o med om he amyloidogenic and he non-amyloidogenic pheno ypes a e dis inc ly di e en om each o he . Dynamic ligh sca e ing (DLS) allows he examina ion o appa en hyd odynamic adius (RH) o a p o ein in solu ion33,34. DLS analysis on he β -oligome s p epa ed unde acidic condi ions show ha H572R β -oligome s exhibi la ge a ia ions in hei RH (~89.95 nm|pH 3.0, ~69 nm|pH 4.5 and ~155 nm|pH 5.5), while R555W β -oligome s a e mo e uni o m and almos simila ac oss a ious acidic pH condi ions (~39.58 nm|pH 3.0, ~51.9 nm|pH 4.5 and ~68.2 nm|pH 5.5). The dis ibu ion cu es om he % in ensi y plo s also show he homogenei y o he non-amyloidogenic R555W, and ela i e he e ogenei y o he amyloidogenic H572R. This is in conjunc- ion wi h he esul s ob ained om TEM, whe e we see mo e homogenous and smalle β -oligome s om he non-amyloidogenic R555W mu an and he e ogeneous and ela i ely la ge β -oligome s om he amyloidogenic H572R mu an . The β-oligome s o he amyloidogenic H572R shows s onge binding o Thio la in T (ThT). Amyloid ib ils bind o he dye ThT and display an emission luo escence a 485 nm35,36. In Alzheime ’s disease, ThT binds o Aβ -oligome s hemsel es, and has been p oposed o ea ly diagnosis37. We wan ed o es i he TGFβ Ip β -oligome s bind o ThT. The amyloid o ming TGFβ Ip pep ide pN622K (pN622K611–633) displayed a high luo escence in ensi y when bound o ThT (Fig.6c). The luo escence displayed by WT was compa able o he backg ound luo escence. Rela i ely, he β -oligome s o H572R showed signi ican luo escence on binding o ThT (**P < 0.01) and a leas ~3 imes mo e luo escence compa ed o R555W. While he luo escence in ensi ies we e much lowe compa ed o he ib il o ming pep ide, i was signi ican ha he β -oligome s o he amyloido- genic H572R we e able o bind o ThT and his could aid u he cha ac e iza ion o he β -oligome s. The mu an s display di e ences in hei ‘agg ega ion ho spo s’. To de e mine he egions wi h high agg ega ion p opensi ies o ‘agg ega ion ho spo s’ wi hin he mu an s and he β -oligome s, he domains we e diges ed wi h ypsin and he esul ing pep ides we e examined using LC-MS/MS. The pep ide map gen- e a ed (Supplemen a y da a) ollowing he insilico ypsin diges ion displayed a se ies o pep ides (Fig.6d) o med ollowing ypsin diges ion. We aimed o iden i y he egions ha we e p obably bu ied wi hin he β -oligome s and hence esis ed ypsin diges ion. Fo WT, h ee sho pep ides (549ALPPR553, 591SLQGDK596 603NNVVSVNK610) we e no de ec able a e yp ic diges ions. Fo R555W, wo sho pep ides (558LLGDAK563, 591SLQGDK596 we e absen . Howe e , o he H572R, pep ides in he egion E611-L632 we e no obse ed in addi ion o a sho pep ide (591SLQGDK596) ha was absen in he WT and R555W. Mapping he gene a ed pep- ides o he 4 h_FAS1 domain displayed in e es ing esul s (Fig.6e). The en i e C- e minal egion encompassing he esidues 603NNVVSVNK610 and 611EPVAEPDIMATNGVVHVITNVL632 pep ides was absen in he H572R na i e p o ein and β -oligome s. This egion was in ac in WT and R555W. A long s e ch o esidues be ween E534 and K563 con aining he pep ides 534EGVYTVFAPTNEAFR548, 549ALPPR553, 554EWSR557, 558LLGDAK563 was no obse ed in R555W β -oligome s. The pep ide 591SLQGDK596 was absen in all he p o eins. The segmen WT R555W H572R Cha ge o he mu a ion –R ( + 1) → W(0) H (0) → R(+1) (a pH 7.0) H (+1) → R(+1) (a pH < 6.0) Ne Cha ge (a pH 7.0) −0.6 − 1.6 0.2 Ne Cha ge (a pH 5.5) 1.9 0.9 2.0 Change in hyd ophobici y due o he mu a ion (17) – 3.95 → − 2.13 (a pH 7.0 and pH < 6.0) 0.64 → 3.95 (a pH 7.0) 2.87 → 3.95 (pH < 6.0) Table 1. E ec s o mu a ion on he cha ge and hyd ophobici y. The change in he indi idual cha ges and o e all ne cha ge in he mu an s. The change in hyd ophobici ies we e calcula ed using he equa ion desc ibed p e iously (17). www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 549ALPPR553 was absen in H572R β -oligome s. The β -oligome s o R555W, displayed a egion ex ending be ween E534 and A562 ha could possibly be bu ied. I is in e es ing o no e ha he mu a ion R555W esides wi hin his egion. The β -oligome s o H572R did no e eal such a egion wi hin he domain. I is likely ha di e en egions o he 4 h_FAS1 domain may be in ol ed in he o ma ion and s abiliza ion o hese β -oligome s. The β-oligome s emained s able a physiological condi ions. Fo s udying he he mal s abili y o he β -oligome s o he R555W (Fig.7a,b) and H572R (Fig.7c,d) he mu an domains a pH 5.5 we e hea ed o 70 °C and cooled back o 20 °C. The β -oligome s emained in hei β -shee con o ma ion a 20 °C showing no e e sibili y o he na i e α /β -con o ma ion. To es hei s abili ies a physiological pH, he β -oligome s o med a pH 5.5 we e econs i u ed o pH 7 and examined (Fig.7e, ). The samples we e also incuba ed o 4 weeks a pH 7 and he CD spec a we e eco ded. In bo h cases, he β -oligome s emained in hei s able β -shee Figu e 2. The mal dena u a ion o he 4 h_FAS1 domains o he WT and mu an s a neu al and basic pH. (a,b) Fa UV CD spec a o he 4 h_FAS1 domain o WT a pH 7.0 and pH 8.0 be o e hea ing (black), a e hea ing o 70 °C ( ed) and cooling back o 20 °C (blue). (c,d) Fa UV CD spec a o he 4 h_FAS1 domains o R555W a pH 7 (c) and pH 8 (d) be o e hea ing (black) and a e hea ing o 70 °C ( ed) and cooling back o 20 °C (blue). (e, ) Fa UV CD spec a o he 4 h_FAS1 domains o H572R a pH 7.0 (e) and pH 8.0 ( ) be o e hea ing (black) and a e hea ing o 70 °C ( ed) and cooling back o 20 °C (blue). The WT and he mu an s did no display any signi ican changes in s uc u e a pH 7.0 and pH 8.0. www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 con o ma ion. This demons a es ha he β -oligome s we e s able o he mal changes and a physiological con- di ions allowing o u he examina ion. Cy o oxici y o β-oligome s. The oxici y o he β -oligome s on p ima y human co neal s omal ib o- blas (pHCSF) cells was moni o ed using xCELLigence sys em. The WT domain did no in e e e wi h he cell adhesion and p oli e a ion and exhibi ed li le o no oxic e ec on he seeded cells (Fig.8a). R555W also dis- played no cy o oxici y. H572R, howe e , displayed highe cy o oxici y (**P < 0.01) on he ib oblas s compa ed o R555W (Fig.8b). In e es ingly, β -oligome s om bo h R555W and H572R we e cy o oxic (**P < 0.01). While, he β -oligome s o R555W dec eased he cell p oli e a ion and he cy o oxic e ec was isible a e 12 hou s, he β -oligome s o H572R displayed he maximum cy o oxic e ec as no p oli e a ion was obse ed and he xCELLigence showed minimum cell index. These esul s sugges ha amyloidogenic mu an displayed signi ican cy o oxic e ec bo h in he na i e as well as in he β -oligome ic o ms whe eas he non-amyloidogenic mu an was cy o oxic in he β -oligome ic o m only. To ob ain de ailed in o ma ion on he cy o oxici ies, we p epa ed he β -oligome s o he wo mu an s a a ious acidic pH (pH 3.0, pH 4.5, pH 5.5). We also included he insoluble Figu e 3. The mal dena u a ion o he 4 h_FAS1 domains o he WT and mu an s a acidic pH. (a–c) Fa UV CD spec a o he 4 h_FAS1 domain o WT a pH 3.0 (a), pH 4.5 (b) and pH 5.5 (c) be o e hea ing (black) and a e hea ing o 70 °C ( ed) and cooling back o 20 °C (blue). (d– ) Fa UV CD spec a o he 4 h_FAS1 domain o R555W a pH 3 (d), pH 4.5 (e) and pH 5.5 ( ) be o e hea ing (black) and a e hea ing o 70 °C ( ed) and cooling back o 20 °C (blue). (g–i) Fa UV CD spec a o he 4 h_FAS1 domain o H572R a pH 3.0 (g), pH 4.5 (h) and pH 5.5 (i) be o e hea ing (black) and a e hea ing o 70 °C ( ed) and cooling back o 20 °C (blue). While he WT did no show any changes in s uc u e, bo h he mu an s displayed a e y clea ansi ion o β -shee unde acidic condi ions. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 agg ega es ob ained a e cen i uging he mildly p ecipi a ed samples. When we examined his p ecipi a es using CD, we ound simila β -shee cu es as we ound o he soluble oligome s (da a no shown). Hence, we in es i- ga ed hese “insoluble agg ega es” along wi h he soluble β -oligome s. The popula ions we e econs i u ed o pH 7 using bu e exchange. pHCSFs om 3 di e en dono s (n = 3) we e ea ed wi h he β -oligome s and examined (Fig.8c). The WT shows almos no cy o oxici y, simila o he con ol. The soluble β -oligome s de i ed om bo h he mu an s a a ious acidic pH condi ions displayed po en cy o oxic e ec (**P < 0.01) compa ed o he insoluble agg ega es and con ols. The insoluble agg ega es we e also cy o oxic (*P < 0.05) bu we e ela i ely lesse compa ed o he soluble β -oligome s. The esul s we e also alida ed using an MTT assay (Fig.8d). Simila o xCELLigence, we could see ha soluble β -oligome s de i ed om bo h he mu an s displayed po en cy o oxic e ec (**P < 0.01) compa ed o he con ols and insoluble agg ega es. Discussion The mos signi ican aspec o he TGFβ Ip associa ed co neal dys ophies is ha he single amino acid subs i- u ions in he mu an s a e esponsible o clinically dis inc pheno ypes. He e, we examined he p ope ies o Figu e 4. Di e ence in he mal dena u a ion induced ansi ion be ween non-amyloidogenic and amyloidogenic mu an s a acidic pH. (a–e) Va iable empe a u e CD cu es a 222 nm o WT (black), R555W ( ed) and H572R (blue) p o eins hea ed om 20 °C o 70 °C a a ious pH (3.0 [a], 4.5 [b], 5.5 [c], 7.0 [d] and 8.0 [e]) and he CD in ensi ies a 222 nm we e plo ed as a unc ion o empe a u e. The baseline sub ac ed cu es o he WT (black), R555W ( ed) and H572R (blue) p o eins show ha while he e was no ansi ion obse ed in he WT in all he condi ions as obse ed om he unchanged s aigh line in black, li le o no changes we e seen in pH 7 and pH 8 o he mu an s. Howe e , clea ansi ions o β -shee we e obse ed a acidic pH (pH 3, pH 4.5 and pH 5.5) o bo h he mu an s. In all cases, we obse e ansi ion (T ) is highe o R555W compa ed o H572R. A clea shi in hei he mal dena u a ion cu es be ween he mu an s a acidic pH (pH 3.0, 4.5 and 5.5) is obse ed. A di e ence in T o 5–12 °C is obse ed a a ious pH condi ions. www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 wo mu an s, he amyloidogenic H572R22,23, and non-amyloidogenic R555W24–26. P e ious a emp s o s udy he di e ences be ween he WT and he mu an s a physiological condi ions ha e e ealed minimal in o ma ion on he mechanism o hei agg ega ion38. To elucida e he inhe en a ia ions be ween hese mu an s, we examined he e ec s o a ious biophysical and biochemical ac o s like empe a u e and pH on hem. In amyloid o ming p o eins such as β -mic oglobulin39, he mal un olding leads o o ma ion o β -oligome s. Resul s om ou he - mal dena u a ion s udies show clea di e ences be ween bo h he pheno ypes and he WT p o ein a acidic pH. Ou esul s sugges ha changes in he physiological pH can in luence he biochemical and biophysical cha - ac e is ics o TGFβ Ip mu an s. A clea con e sion om α /β -s uc u e o β -shee was obse ed when he mu an s we e hea ed unde acidic condi ions (< pH 5.5). TEM and DLS s udies con i med he o ma ion o β -oligome s by bo h pheno ypes ha emain i e e sible and s able a physiological pH and empe a u e. Fo he i s ime we ha e shown ha bo h he amyloidogenic and non-amyloidogenic pheno ypes displayed ma ked di e ences Figu e 5. pH sensi i i y and s abili y o he non-amyloidogenic (R555W) pheno ype. (a) Fluo escence emission spec a o R555W wi h dec ease in pH. The e was a clea dec ease in emission maximum a 332 nm wi h dec ease in pH (indica ed by he black a ow). (b–e) Fluo escence emission spec a o R555W showing he e e sibili y o olded s a e a e emo al o u ea. The R555W mu an was incuba ed wi h inc easing concen a ions o u ea om 0.25 M o 8 M a a ious acidic condi ions (pH 3, pH 4.5, pH 5.5) and pH 7 and he emission luo escence be o e and a e u ea incuba ion was measu ed. The emission spec a be o e u ea incuba ion 332 nm (black), a e incuba ing wi h 8 M u ea ( ed) and a e emo ing u ea by bu e exchange (blue). Un olding o he p o ein is seen by he shi ing o peaks (black a ow) om 332 nm o ~ 352 nm. The e olding o he p o ein a e emo al o u ea is seen by he e u n o he emission maximum o ~332 nm (g een a ow). ( –i) In es iga ion o he s abili y he non-amyloidogenic pheno ype using u ea dena u a ion s udies. The R555W mu an was incuba ed wi h inc easing concen a ions o U ea om 0.25 M o 8 M a a ious acidic pH (pH 3, pH 4.5, pH 5.5) and pH 7, and he emission luo escence was measu ed. The dena u a ion plo s o ‘ ac ion un olded s u ea concen a ion’ we e plo ed and i in o a wo s a e model, wi h he pa ame e s calcula ed as desc ibed in he me hods sec ion. pH Δ G H 2O Z, kJ/mole Cm, M m, kJ/mole/M ΔΔGH2O , kJ/mole 3.0 8.417 ± 1.03 4.2 ± 0.15 3.14 ± 0.32 − 5.383 4.5 7.108 ± 1.5 4.74 ± 0.91 1.85 ± 0.25 − 6.692 5.5 11.28 ± 2.02 3.93 ± 0.21 2.78 ± 0.48 − 2.52 7.0 13.8 ± 0.8 3.09 ± 0.1 3.8 ± 0.3 – Table 2. U ea dena u a ion s udies o he non-amyloidogenic pheno ype. The R555W 4 h FAS1 domain was incuba ed wi h inc easing concen a ions o u ea om 0.25 M-8 M a a ious acidic pH condi ions and he emission luo escence was measu ed. The ee ene gies a a ious pH condi ions we e calcula ed and abula ed. www.na u e.com/scien i ic epo s/ 9 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 Figu e 6. Cha ac e iza ion amyloidogenic and non-amyloidogenic β-oligome s. (a–b) T ansmission Elec on Mic oscopy. TEM images o he β -oligome s o he 4 h_FAS1 domains o R555W (a) and H572R (b) mu an s we e acqui ed wi h a JEOL JEM-1010 ansmission elec on mic oscope using Digi al Mic og aph™ 1.81.78 o GMS 1.8.0. The β -oligome s o he amyloidogenic pheno ype we e la ge measu ing be ween 10–40 nm, mean size ~19.1 nm ± 4.9 nm (a) compa ed o he non-amyloidogenic β -oligome s ha measu ed 4–8 nm, wi h a mean size ~5.1 nm ± 1.79 nm (b). Inse igu es – pa icle size dis ibu ion o he β -oligome s. www.na u e.com/scien i ic epo s/ 16 Scien i ic RepoR s | 6:23836 | DOI: 10.1038/s ep23836 13. Lakshmina ayanan, R. e al. S uc u e-Func ion Rela ionship o TGFβ Ip (BIGH3) Associa ed Mu an s in Co neal Dys ophy. B i . J. Oph halmol. 95(10), 1457–62 (2011). 14. Ołdak, M. e al. La e-onse la ice co neal dys ophy wi hou ypical la ice lines caused by a no el mu a ion in he TGFBI gene. Co nea 33(3), 294–9 (2014). 15. Klin wo h, G. K., Valnicko a, Z. & Enghild, J. J. 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Linea ex apola ion me hod o analyzing sol en dena u a ion cu es. P o eins Suppl 4, 1–7 (2000). 60. U can, E. e al. Real- ime xCELLigence impedance analysis o he cy o oxici y o den al composi e componen s on human gingi al ib oblas s. Den al Ma e ials 26, 51–58 (2010). Acknowledgemen s The au ho s would like o hank D . Ga y Yam o his help ul insigh s. The p ojec was unded by NMRC Singapo e (CS-IRG g an R978/87/2012). Au ho Con ibu ions E.M., L.R. and J.S.M. concei ed he expe imen s and w o e he pape . E.M. cloned he genes, exp essed he p o eins, pe o med he CD luo escence, TEM expe imen s and p epa ed he oligome s o MS expe imen s and cy o oxici y s udies. A.V., V.M. and N.M. assis ed in he exp ession and pu i ica ion o he p o eins. Z.L. pe o med he MS expe imen s. N.M. and E.G. assis ed wi h he CD expe imen s on he mal dena u a ion. R.R.Y.L., G.S.L.P. and S.S.C. pe o med he cy o oxici y assays. R.W.B. and S.S.C. p o ided inpu s o he concep ion o he wo k and edi ing he a icle. Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/s ep Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s. How o ci e his a icle: Mu ugan, E. e al. pH Induced Con o ma ional T ansi ions in he T ans o ming G ow h Fac o β-Induced P o ein (TGFβIp) Associa ed Co neal Dys ophy Mu an s. Sci. Rep. 6, 23836; doi: 10.1038/s ep23836 (2016). This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in he c edi line; i he ma e ial is no included unde he C ea i e Commons license, use s will need o ob ain pe mission om he license holde o ep oduce he ma e ial. To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/