- Email: [email protected]
Glycation of γ crystallin and lens opacification
Thursday, 5:30-7:00 P.M., Sep 24, 1992 Palazzo Dei Congressi
X ICER Abstracts 679
49 TRANSMCATION SHOCK IN CLONAL
AND JNDUCI’ION GLIOMA CELJS
OF aB CRYSTALLIN
Department of Biochcmishy, Institute forDc.velopmental Prefectural Colony, Kasugsi, Aicbi, Japan
ACTIVATION OF BOVINE LENS ALDOSE REDUCTASE BY CARBOXYMETHYLATION Srivastava. S.K.. Liu. S-O.. Bhatnaaar. A.. and Ansari. N.H. Department of Human Biological Chemistry 6 Genetics, University of Texas Medical Branch, Galveston, Texas, U.S.A.
BY HEAT
Reseamh, Aicbi
Bovine lens aldose reductase (AR) purified to homogeneity was reduced by 10 mM dithiothreitol (DTT) and filtered through Sephadex G-25 column to remove free DTT. Incubation of completely reduced aldose reductase with 10 ti iodoacetate for 1 hr leads to a more when the enzyme activity was determined than 4 fold increase ink,., in 75 mN potassium-phosphate, pH 7.0. 1 mll EDTA, 10 mM DLelvceraldehvde and 0.1 mM NADPH. Carboxvmethvlation also results in 3-5 fold increase in K, NADPH and % D.g21cPralAehydo, whereas K,,, L. Blycsraldehyde increases approximately 30 fold. The carboxymethylated enzyme becomes less sensitive to inhibition by 5,5'-dithiobis (2. nitrobenzoic acid), Sorbinil (K,, increased from 0.4 to 109 @M) and NADP (K,, increased from 0.01 to 0.03 mM) but not by tolrestar. This differential effect of sorbiniland tolrescat suggests atleast two inhibitor binding sites, an 'S' site for sorbinil and a 'T' site for tolrestat. Activation by IAA was prevented by NADPH. Hydride transfer as well as substrate binding steps are not significantly affected by carboxymethylacion. The increased k,,,, could be due to facilitation of the isomerization of the E:NADP binary complex. Carboxymethylation-induced changes in the human placental AR are similar to bovine lens enzyme.
Responses to heat shock of aB crystallin and small heat shock protein (HSP28) in culture cells wen analyzed quantitatively by the use of immunoassay methods. When rat glioma GA-I cells, expressing aB crystallin, were heated at 45°C concentrations of aF3 crystallin in cell extracts d-sed to less than one-fifth of the original level within 15 min. with an increase in the insoluble fraction which was detected by immunoblotling. The low level of nB mystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the cona level within several hours. At 10 h following heat shock (4X for 15 mitt), the concenuation of aB crysudlin in the soluble extract was about twice that of the contml level, with little detectable amounts in the insoluble fraction. Human glioma U373 cells. producing considerable levels of aB crystallin and human HSP28, were subjected to beat shock as described above. Concenuations of bolh aB crystallin and HSP28 in the extract of U373 cells displayed changes similar to those of aB crystallin observe in GA-I cells against heat shock. These resulrs are additional evidences that suggest that aB crystallin is a small heat shock protein.
I
1
660
50 aB-CRYSTALLIN SUBSTRATES Department Netherlands
I,
AND 6-CRYSTALLINS AS FOR TISSUE TRANSGLUTAMINASE
of Biochemistry,
University
of Nijmegen,
MOLECULAR
AMINE-DONOR
National
Nijmegen,
The
WEIGHT
DISTRISUTION
Research
Institute
OF oCRYSTALLlN
of Australia,
Carlton,
IN SINGLE BOVINE Victoria
3053,
LENSES
AUSTRALIA
The quaternary mxtum of ocrystallin has long bow tha subiect of controvarsv. It iabdiavedbvaome,thatthaprotdnhasathrea+amdefrsnpmn t of subunits. In the young lans, it is anvisag~I that tha outer two hs am y&timua$ fill *s tha Ian8 agas. lf tbiMs is d-18 caw, dasptta B contrary, it heuld k pcMibb to isdam gmtb of isffmmm a$w wbieh havs diiemm tttdeeubr vwifpta, differem subudt wnmma WA dirtrtan microenvironmmts for spa&c amino acids. Tha Present studv examines this possibility.
The aminedonor substrati for transglutaminase among lens proteins were identified, using a biotinylated amine-acceptor peptide as a prohe. The probe was mcdelled on the sequence around the amine-acceptor glutamine residue in bovine BA3-crystallin. Ca’+-activated transglutaminase linked this amine-acceptor peptide to several Bcrystallins, but also to aE-crystallin. To identify the site of linkage, bovine arB-aystallin was digested with carhoxypeptidase B. Subsequent streptavidin blot analysis showed that aB does not react with the amineacceptor pro& upon removal of the C-terminal lysine residue. This was also found for aB-crystallin from dogfish and chicken lens, which have a C-terminal lysine at comsponding positions. These results demonstrate that this conserved C-terminal lysine serves as aminodonor substrate for transglutaminase in aB-cryStallin. This strengthens the notion that, at least for crystallins, all transgl~inaae substrate residues are located in terminal extensions of the polypeptides. In lens homogenates, olBcrystallin can he covalently li&al to B-crystallins by transglutaminase. Transglutaminasc-mediated crosslinking of aB-crystallin outside the lens, could have biological implications for the role of aB-crystallin in nourodegenerative disorders or in heart ischemia.
The lens has a unique studving~P-ofdevrlopoM
dcwbpmantal
lenses into f seriw of direct comparisons batwaen
proteins
pattern thal mdtaa A a” mde&ng.Nmkmres&gmwontcqtof
ideal
lavorr. 9tudba of ttwat kiwi* lHows of diff@sM agu from the earna Isns.
tisnue
for
&ailed,
a-Crystallin ‘was pudfii from each taper, isolated from adult 129) and foatal (0.791 bovine lenra, by HfI’Z gal fikrltbn u&t9 a SuprOer 12 cokrfnn. MdecU18, wdgtltswwcdnsnnhnd uQlotluM~~aeasubg.Tbsywswfoundto increa6e~fnwnthepaiphsryBr~cenmot~adukhn+,fmmabovt500 kDa up fo about 3ODO kD& Cham in tha molecular wai&t of tha protein from foetal lenses wan much less pronounced.
microenvironmmts illmmMtowardathwmmef~lmc.liow#er,Ih~wa5m ‘et*?-ewdencs or~ducslof~plOupo.A#~~ocartrirnmwkhssingle class of SH groups, of which ab+ut 10% bacorne axposad We musf concfude,
again,
that u.crystallin
cannot
ss the protein
have a three-layered
ages. structura.
661
51 GLYCATION OF y CRYSTALLIN AND LENS OPACIFICATION Pande. Aiay Boston Biomedical Research Institute, Boston, Massachusetts,
Vision
THE SUBMIT COMPDSfTlON OF 4RYSTALLIN DURING AGEING S&w-m A. mdBuprppm,&(; National Vision Research Institute, Carfton, Victoria, AUSTRALIA
USA
a-Crystallin, tha major structural protein of the ocukir lans is composed of twa subunits, aA and aB. Their ratio is known to vary in dfffuam apaeias but there are confliing rcqmts as to the changas that occur with ag&ng and devdopment. In bovkmbnsss,rqmrtadA:Bvakmsraqahanaslowas2:1 toahighof4:l. depandingontiageofthalens. lthasalsotm8nrapormdthattharatiochangw during difMm&ion fmm around 1:2 in tha epithMd layers to 3:l in tha nucleus fibra cells. If &a ratio changea with d+v&pmam than it shaufd ba po#sM to isdsh,~vffkus~~anlidsntify~~uafunctknofrgdng. Several dtffnan opproscher Wfw uS0d 03 isdns isoforms d eeqst&in, including amity elwv of cllnmhls. D ~vhv on DEAE-ca!.luloreandisdalkmofcrcrytgllinsof~ppasfromanwnnk:hyars from the 10mb lens. subunr ratios wwe #btafhW with SDS-PAGE. !iSnes the lvsins contenta of tfm two subunits diffar, calWr8tkm g&s ware fiat construct& to datannine whether a corm&m for l~sine contant Jane wa s&i&ant to &mate the subunit rat6o in tha diffwant isoforms. Tha SDS @s e thaw a furthar correction is rag&red to take into account the non-w obwrvrd, tica * ratio of added subunits was Mft‘twlt from that obsal-4 u&g dana&xmmic scamli~. The results with whole bnsas indicmd that thm is only ona isoform of a-crystalfin with a subunit r&o of 3A to 16 chain. BY contrast, uraa-traared o-crvstallin consisted of isoforms with ratios varYin from 2.2~5.3:1. lonaxehange of crosslinkad a-aystallin rewmed in ona pa& and data&d chnnctarix&on of the subunit comp&s&w across ti paak mw&d a -m rubunit ratio furdwr suppcrdng the view Illat only one iwform of me pratdn Mists in ti lens. ocrVst&lins irohied from conwntdc lakers nmowd from a *Ia fti bs alho contained a fixed subunit ratio of 3:1 for moat of the lansnr. The @U&MI Lamar had a slightly lowr rat& aluwhg th~viawtfmtlaleindav&opmmttofthelans,tie svnthesis of A:0 chain8 changes and mom S chains are qnthesized than A chains.
Glycation (or non-enzymatic glycosylation) of proteins is an important post-translational modification in tissues such as the lens where protein turnover is negligible. Over time, the concentration of the glycated proteins gradually increases to very high levels and alters the tissue dramatically. We have investigated the effect of glycation on lens y crystallins, since these are the longesblived lens proteins. As a model system, the in vitro reaction between calf $1 crystallin, and fructose was studied. The reaction leads to the formation of a soluble and an insoluble protein fraction. Of the total soluble protein, about 50% is present as non-reducible, covafently crosslinked aggregates. The critical temperature for phase separation (i.e. Tc) of the soluble fraction increases by about 15°C compared to the native 711 solution. However, no evidence of a conformational change is observed, as deduced from far-u.v. circular dichroism. The formation of large protein aggregates and an increase in Tc, resulting from the glycation of y crystallin solutions, are precisely the conditions favoring lens opacification. It is likely that similar changes occur in other post-translational modifications of the ycrystallins, and protein-unfolding may not be a prerequisite for opacification.
s.197
X ICER Abstracts 679
49 TRANSMCATION SHOCK IN CLONAL
AND JNDUCI’ION GLIOMA CELJS
OF aB CRYSTALLIN
Department of Biochcmishy, Institute forDc.velopmental Prefectural Colony, Kasugsi, Aicbi, Japan
ACTIVATION OF BOVINE LENS ALDOSE REDUCTASE BY CARBOXYMETHYLATION Srivastava. S.K.. Liu. S-O.. Bhatnaaar. A.. and Ansari. N.H. Department of Human Biological Chemistry 6 Genetics, University of Texas Medical Branch, Galveston, Texas, U.S.A.
BY HEAT
Reseamh, Aicbi
Bovine lens aldose reductase (AR) purified to homogeneity was reduced by 10 mM dithiothreitol (DTT) and filtered through Sephadex G-25 column to remove free DTT. Incubation of completely reduced aldose reductase with 10 ti iodoacetate for 1 hr leads to a more when the enzyme activity was determined than 4 fold increase ink,., in 75 mN potassium-phosphate, pH 7.0. 1 mll EDTA, 10 mM DLelvceraldehvde and 0.1 mM NADPH. Carboxvmethvlation also results in 3-5 fold increase in K, NADPH and % D.g21cPralAehydo, whereas K,,, L. Blycsraldehyde increases approximately 30 fold. The carboxymethylated enzyme becomes less sensitive to inhibition by 5,5'-dithiobis (2. nitrobenzoic acid), Sorbinil (K,, increased from 0.4 to 109 @M) and NADP (K,, increased from 0.01 to 0.03 mM) but not by tolrestar. This differential effect of sorbiniland tolrescat suggests atleast two inhibitor binding sites, an 'S' site for sorbinil and a 'T' site for tolrestat. Activation by IAA was prevented by NADPH. Hydride transfer as well as substrate binding steps are not significantly affected by carboxymethylacion. The increased k,,,, could be due to facilitation of the isomerization of the E:NADP binary complex. Carboxymethylation-induced changes in the human placental AR are similar to bovine lens enzyme.
Responses to heat shock of aB crystallin and small heat shock protein (HSP28) in culture cells wen analyzed quantitatively by the use of immunoassay methods. When rat glioma GA-I cells, expressing aB crystallin, were heated at 45°C concentrations of aF3 crystallin in cell extracts d-sed to less than one-fifth of the original level within 15 min. with an increase in the insoluble fraction which was detected by immunoblotling. The low level of nB mystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the cona level within several hours. At 10 h following heat shock (4X for 15 mitt), the concenuation of aB crysudlin in the soluble extract was about twice that of the contml level, with little detectable amounts in the insoluble fraction. Human glioma U373 cells. producing considerable levels of aB crystallin and human HSP28, were subjected to beat shock as described above. Concenuations of bolh aB crystallin and HSP28 in the extract of U373 cells displayed changes similar to those of aB crystallin observe in GA-I cells against heat shock. These resulrs are additional evidences that suggest that aB crystallin is a small heat shock protein.
I
1
660
50 aB-CRYSTALLIN SUBSTRATES Department Netherlands
I,
AND 6-CRYSTALLINS AS FOR TISSUE TRANSGLUTAMINASE
of Biochemistry,
University
of Nijmegen,
MOLECULAR
AMINE-DONOR
National
Nijmegen,
The
WEIGHT
DISTRISUTION
Research
Institute
OF oCRYSTALLlN
of Australia,
Carlton,
IN SINGLE BOVINE Victoria
3053,
LENSES
AUSTRALIA
The quaternary mxtum of ocrystallin has long bow tha subiect of controvarsv. It iabdiavedbvaome,thatthaprotdnhasathrea+amdefrsnpmn t of subunits. In the young lans, it is anvisag~I that tha outer two hs am y&timua$ fill *s tha Ian8 agas. lf tbiMs is d-18 caw, dasptta B contrary, it heuld k pcMibb to isdam gmtb of isffmmm a$w wbieh havs diiemm tttdeeubr vwifpta, differem subudt wnmma WA dirtrtan microenvironmmts for spa&c amino acids. Tha Present studv examines this possibility.
The aminedonor substrati for transglutaminase among lens proteins were identified, using a biotinylated amine-acceptor peptide as a prohe. The probe was mcdelled on the sequence around the amine-acceptor glutamine residue in bovine BA3-crystallin. Ca’+-activated transglutaminase linked this amine-acceptor peptide to several Bcrystallins, but also to aE-crystallin. To identify the site of linkage, bovine arB-aystallin was digested with carhoxypeptidase B. Subsequent streptavidin blot analysis showed that aB does not react with the amineacceptor pro& upon removal of the C-terminal lysine residue. This was also found for aB-crystallin from dogfish and chicken lens, which have a C-terminal lysine at comsponding positions. These results demonstrate that this conserved C-terminal lysine serves as aminodonor substrate for transglutaminase in aB-cryStallin. This strengthens the notion that, at least for crystallins, all transgl~inaae substrate residues are located in terminal extensions of the polypeptides. In lens homogenates, olBcrystallin can he covalently li&al to B-crystallins by transglutaminase. Transglutaminasc-mediated crosslinking of aB-crystallin outside the lens, could have biological implications for the role of aB-crystallin in nourodegenerative disorders or in heart ischemia.
The lens has a unique studving~P-ofdevrlopoM
dcwbpmantal
lenses into f seriw of direct comparisons batwaen
proteins
pattern thal mdtaa A a” mde&ng.Nmkmres&gmwontcqtof
ideal
lavorr. 9tudba of ttwat kiwi* lHows of diff@sM agu from the earna Isns.
tisnue
for
&ailed,
a-Crystallin ‘was pudfii from each taper, isolated from adult 129) and foatal (0.791 bovine lenra, by HfI’Z gal fikrltbn u&t9 a SuprOer 12 cokrfnn. MdecU18, wdgtltswwcdnsnnhnd uQlotluM~~aeasubg.Tbsywswfoundto increa6e~fnwnthepaiphsryBr~cenmot~adukhn+,fmmabovt500 kDa up fo about 3ODO kD& Cham in tha molecular wai&t of tha protein from foetal lenses wan much less pronounced.
microenvironmmts illmmMtowardathwmmef~lmc.liow#er,Ih~wa5m ‘et*?-ewdencs or~ducslof~plOupo.A#~~ocartrirnmwkhssingle class of SH groups, of which ab+ut 10% bacorne axposad We musf concfude,
again,
that u.crystallin
cannot
ss the protein
have a three-layered
ages. structura.
661
51 GLYCATION OF y CRYSTALLIN AND LENS OPACIFICATION Pande. Aiay Boston Biomedical Research Institute, Boston, Massachusetts,
Vision
THE SUBMIT COMPDSfTlON OF 4RYSTALLIN DURING AGEING S&w-m A. mdBuprppm,&(; National Vision Research Institute, Carfton, Victoria, AUSTRALIA
USA
a-Crystallin, tha major structural protein of the ocukir lans is composed of twa subunits, aA and aB. Their ratio is known to vary in dfffuam apaeias but there are confliing rcqmts as to the changas that occur with ag&ng and devdopment. In bovkmbnsss,rqmrtadA:Bvakmsraqahanaslowas2:1 toahighof4:l. depandingontiageofthalens. lthasalsotm8nrapormdthattharatiochangw during difMm&ion fmm around 1:2 in tha epithMd layers to 3:l in tha nucleus fibra cells. If &a ratio changea with d+v&pmam than it shaufd ba po#sM to isdsh,~vffkus~~anlidsntify~~uafunctknofrgdng. Several dtffnan opproscher Wfw uS0d 03 isdns isoforms d eeqst&in, including amity elwv of cllnmhls. D ~vhv on DEAE-ca!.luloreandisdalkmofcrcrytgllinsof~ppasfromanwnnk:hyars from the 10mb lens. subunr ratios wwe #btafhW with SDS-PAGE. !iSnes the lvsins contenta of tfm two subunits diffar, calWr8tkm g&s ware fiat construct& to datannine whether a corm&m for l~sine contant Jane wa s&i&ant to &mate the subunit rat6o in tha diffwant isoforms. Tha SDS @s e thaw a furthar correction is rag&red to take into account the non-w obwrvrd, tica * ratio of added subunits was Mft‘twlt from that obsal-4 u&g dana&xmmic scamli~. The results with whole bnsas indicmd that thm is only ona isoform of a-crystalfin with a subunit r&o of 3A to 16 chain. BY contrast, uraa-traared o-crvstallin consisted of isoforms with ratios varYin from 2.2~5.3:1. lonaxehange of crosslinkad a-aystallin rewmed in ona pa& and data&d chnnctarix&on of the subunit comp&s&w across ti paak mw&d a -m rubunit ratio furdwr suppcrdng the view Illat only one iwform of me pratdn Mists in ti lens. ocrVst&lins irohied from conwntdc lakers nmowd from a *Ia fti bs alho contained a fixed subunit ratio of 3:1 for moat of the lansnr. The @U&MI Lamar had a slightly lowr rat& aluwhg th~viawtfmtlaleindav&opmmttofthelans,tie svnthesis of A:0 chain8 changes and mom S chains are qnthesized than A chains.
Glycation (or non-enzymatic glycosylation) of proteins is an important post-translational modification in tissues such as the lens where protein turnover is negligible. Over time, the concentration of the glycated proteins gradually increases to very high levels and alters the tissue dramatically. We have investigated the effect of glycation on lens y crystallins, since these are the longesblived lens proteins. As a model system, the in vitro reaction between calf $1 crystallin, and fructose was studied. The reaction leads to the formation of a soluble and an insoluble protein fraction. Of the total soluble protein, about 50% is present as non-reducible, covafently crosslinked aggregates. The critical temperature for phase separation (i.e. Tc) of the soluble fraction increases by about 15°C compared to the native 711 solution. However, no evidence of a conformational change is observed, as deduced from far-u.v. circular dichroism. The formation of large protein aggregates and an increase in Tc, resulting from the glycation of y crystallin solutions, are precisely the conditions favoring lens opacification. It is likely that similar changes occur in other post-translational modifications of the ycrystallins, and protein-unfolding may not be a prerequisite for opacification.
s.197