Photochemistry and PhotobioIogy, Val. 57, No. 4, pp. 739-743, 1993 Printed in the United States. All rights reserved 003 1-8655193 %05.00+O.OC

0 1993 American Society for Photobiologq

RESEARCH NOTE

ALTERATION OF DYNAMIC QUATERNARY STRUCTURE AND CALCIUM-BINDING ABILITY OF &CRYSTALLIN BY LIGHT

YOGENDRA SHARMA, A. GOPALA KRISHNA and D. BALASUBRAMANIAN* Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India

(Received 17 April 1992; accepted 17 July 1992)

Abstract-&crystallin, one of the three main constituent proteins of the eye lens, exists as an equilibrium population of oligomeric (OH), trimeric WL1) and dimeric (pL2) species. This equilibrium is dependent on various factors such as the protein concentration, ionic strength and pH of the medium. We have studied the effect of ultraviolet B radiation on the aggregational patterns of @-crystallin, using size-exclusion chromatography. Irradiation of a solution of @H-crystallin at 295 nm for about 30 min causes the deaggregation of the hexameric population into dimers. Irradiation for a longer time, however, produces cross-linked high molecular weight products. Irradiation of a OL2 solution for 30 min does not perturb the elution profile, while irradiation for a longer time increases the content of BL1 (trimeric) crystallin. Irradiation also causes a decrease in the calcium-binding affinity of the P-crystallins.

INTRODUCTION

Considerable attention has been drawn in recent years to the photochemical and photophysical changes of the eye lens and its protein constituents. Since the eye lens is continuously exposed to light, photodamage is thought to be a factor con- tributing to cataract formation.’-3 This problem has taken on greater urgency due to the fact that (1) the tropical region receives higher doses of the damaging ultraviolet (UV)-B (280-320 nm)t radiation than the temperate zone^^,^ and (2) the incidence of cataract is fourfold higher in these regions of the world.6 Photooxidation of the crystallins, which con- stitute about 40% of the lens weight, is found to induce pro- tein c ro~s - l ink ing ,~~~ increase non-Trp fluorescencePJ0 and cause conformational changes in lens cry stall in^,"-^^ which might alter the interaction of crystallins and lead to changes in packing of the proteins in the lens fiber cells and of the fiber cells themselves, Among the proteins of the mammalian eye lens, a- and P-crystallins are multisubunit structures. Changes that occur in their quaternary structural organiza- tion would be of importance to the way in which they are packed in the lens. We report results on the UV-B-induced changes in the quaternary structural features in this com- munication. The results on a-crystallin will be reported else- where.

P-crystallins are made up of several related basic and acidic subunits that combine to form differently sized oligomers ranging from 50 kDa to 200 kDa. There are at least seven subunits-the basic proteins PBl, PB2, PB3 and the acidic

PAl, PA2, PA3 and PA4 with apparent molecular weights of 33, 26, 26, 22, 22, 26 and 22 kDa, re~pectively.’~ Although some of these subunits are common in all aggregates, PBl is restricted to the aggregate called j3 heavy or PH.15 PB2 (earlier called PBp), which is the principal and common constituent of all P-crystallins, associates with itself or with other mono- mers to yield higher molecular weight aggregates.’”’8 Many studies have dealt with the dynamic quaternary assembly of P-crystallins, which form several aggregated P-crystallins, mainly oligomer (PH), trimer (pL1) and dimer (PL2). How- ever, the quaternary form of these proteins in the lens is not clearly known. In solution, the quaternary structure depends on various factors such as the presence of salts, ionic strength, pH of the mediumIY and the protein concentration.20

The calcium ion concentration is often seen to be altered in human cataractous lenses.2’ The only major molecular species that displays calcium-binding ability in mammalian lenses is P-crystallin, which binds 3 4 mol of calcium per mol with an affinity of0.38 mM.22 The two other mammalian crystallins, a- and y-crystallin, do not do so, while 6-crys- tallin, the core protein in avian and reptilian lenses, does; hence the classification of P- and krystallins as calcium- binding crystallins.” Although P-crystallin is known to be involved in the binding of calcium ions, very little is under- stood about its role in the cataractogenesis. In this work we wish to report changes in the dynamic quaternary structure and calcium-binding ability of bovine P-crystallin upon pho- todamage.

*To whom correspondence should be addressed. ?Abbreviations: PH, hexameric beta-crystallin; pL1, trimeric beta-

crystallin; OL2, dimeric beta-crystallin; CD, circular dichroism; HMW, high molecular weight; LMW, low molecular weight; MOPS, 3-[N-morpholino]propanesulfonic acid UV-B, ultraviolet B ra- diation (280-320 nm).

MATERIALS AND METHODS

Chromatography. Crystallins from bovine lenses were purified on a Bio-Gel A 1.5 m column. Size-exclusion chromatography was done on a Pharmacia fast protein liquid chromatography (F’PLC) system using a Superose 12 gel filtration column in 50 mM Tris buffer containing 50 mM NaCI, pH 7.2.

719

740 YOGENDRA S H A R ~ ~ A et al.

Irradiation. Irradiation of the protein solutions was done in a Hitachi F-4000 fluorescence spectrophotometer using the 150 W Xe arc lamp as the source, with the monochromator set at 295 nm and with an excitation slit width of 20 nm, and with constant stimng of the solution. The amount of light falling on the sample under these conditions was estimated (using both actinometry and light meter) to be of the order of lOI4 photons/s. Aliquots were taken out at the specified time intervals. Fluorescence spectra were recorded using the same instrument.

Circular dichroism (CD) spectroscopy. Circular dichroism spectra were recorded on a JASCO-J2O spectropolarimeter. The solution of the calcium ion mimic dye Stains-all was prepared in 2 mM 3-[N- morpholino]propanesulfonic acid (MOPS) containing 30% ethylene glycol, pH 7.2 as earlier.24 The experiments were performed either in darkness or in as low levels of light as possible to avoid bleaching of the dye. Stains-all was obtained from Sigma Chemical Co., St. Louis, MO. All other chemicals used were of analytical grade.

RESULTS AND DISCUSSION

Effect of light on the quaternary structure of P-crystallin

Quaternary structural changes in a protein are accompa- nied by hydrodynamic changes, which are readily monitored by size-exclusion chromatography. We have, therefore, used this method to monitor the changes in the aggregational sta- tus of P-crystallins. Upon chromatography of a solution of PH-crystallin of concentration greater than 2 mg/mL, five distinct peaks and shoulders are seen, indicating five different populations. The very first peak corresponds to high molec- ular weight (HMW) aggregates, the next is the major and distinct peak for PH corresponding to about 160 kDa (hex- amer), while the next distinct peak corresponds to the dimeric PL2-crystallin. The resolution ofthe trimeric 8-crystallin UL1) depends upon the concentration of PH-crystallin used; at protein concentrations lower than 1 mg/mL, the pL2 content (peak 4) is higher than that of the peak 2 (OH); and an in- termediate shoulder 3 corresponding to PLl is seen. We have, in this study, chosen two different concentrations of pH (2.4 and 0.9 mg/mL). The optimum ionic strength required for the formation of stable aggregates in solution is around 50 m M Tris-HC1 containing 50 m M NaCl at pH 7.5 as is used in this work.

Irradiation in the UV region is known to cause the oxi- dation of the labile amino acid side chains in proteins, e.g. Trp, to various photoproducts and also to perturb the sec- ondary and tertiary structures of the cry stall in^.^^-^^ How- ever, the effect of such irradiation on the quaternary structure or the aggregation profiles of these proteins has not been well studied, and we focus attention on this aspect here. Figure 1A shows that the various aggregational states of this mol- ecule are easily visualized with Superose 12 column chro- matography-peak 1 being the HMW aggregates, peak 2 the PH or hexamer, peak 3, which is seen essentially as a shoulder being the trimer PLl, peak 4 the dimer pL2 and the later peak collectively due to the low molecular weight (LMW) components. Irradiation at 295 nm for 30 min alters the relative abundances of the various fractions. The PH content decreases while the dimeric population (peak 4, pL2) in- creases concomitantly. Prolonged irradiation also leads to an increase in the HMW fractions, again at the cost of the PH fraction.

Figure 1B shows that similar results are obtained with a more dilute solution of the protein (0.9 mg/mL). The trimeric BLl is a little better resolved at this concentration. while the

0 ID N a

~

0 1 . .

I 1 8 12 16 2

VOLUME (rnl) Figure 1. Size exclusion chromatographic profile of the normal and irradiated OH-crystallin on Superose 12 column in 50 mMTris buffer containing 50 mM NaCI, pH 7.5. @-crystallin was irradiated at 295 nm for 0 h (-), for 30 min (.....), for 1 h (---) and for 2 h (-.-.-). Concentration of the protein used was 2.4 mg/mL in panel A and 0.9 mg/mL in panel B. Analyses of the various P-crystallin aggregates were performed by gel filtration using a Pharmacia LKB F'F'LC system equipped with a Superose 12 HR column (1 x 30 cm) eluted at 0.4 mL/min. Sample volumes of 200 fiL were injected and

elution was monitored at 280 nm.

proportion of PH is far smaller here than in Fig. 1A. Irra- diation leads to significant changes in the aggregational profile or the quaternary structure of P-crystallin. The PH and pLI species appear photovulnerable and transform initially intc the dimer pL2 and upon prolonged irradiation into HMN products as well. It is clear that the dissociation into pL: occurs as a prelude to the eventual formation of HMW prod. ucts.

The aggregational status of the dimeric pL2 by itself ap- pears relatively unaffected by light. Figure 2 shows this ver) effectively. Here we see that PL2-crystallin (dimeric form) ir eluted as a major peak (peak 2) along with a minor peak (peak 1) for pL1 -crystallin (trimeric form). Upon irradiatior for about an hour or more, there is a slight increase in the content of trimeric PLI-crystallin, while irradiation for 3C min or less does not cause any significant changes in the elution profile.

Figure 3 shows the changes that occur in the CD spectra of PH-crystallin in the far-UV region (indicative of the sec- ondary structural status of the protein) and in the near-UV region (tertiary structure) upon irradiation for varying peri- ods of time at 295 nm. There are perceptible changes in the tertiary structure (near-UV CD spectra) of the protein within 30 rnin of irradiation (Fig. 3A), particularly in the tryptophan spectral band region near 290 nm. In contrast, the secondar) structural changes seen in the far-UV CD spectra (Fig. 3B) occur only after extensive irradiation (2 h or more). It woulc thus appear that the slight tertiary structural alteration thai occurs upon irradiation is sufficient to trigger a reorganizatior of the quaternary structure and aggregational status of OH- and BL1 -crvstallins.

Research Note 74 1

0 OD RI a

VOLUME (ml 1 Figure 2. Size exclusion chromatographic profile of the normal and irradiated j3L2-crystallin on the Superose 12 column. Sample was irradiated at 295 nm for (---) 0 h, (.....)1 h, (- - -) 2 h, (-.-.-) 4 h. The protein concentration used was 0.79 mg/mL. All

other conditions were as described in the legend to Fig. 1.

We have also noted (data not shown) that the tryptophan fluorescence peak intensity ofp-crystallin drops steadily upon irradiation and that the broad peak around 410 mn due to the photoproducts (N-formylkynurenine and related com- pounds) increases in intensity. Whereas P-crystallin shows all the abovesaid changes, y-crystallin simply precipitates out upon being irradiated for long periods of time. a-crystallin, however, does not show much change.

Analysis of the composition of bovine PH-, PLI - and pL2- crystallins has revealed that the monomer termed PBl is specifically found in PH- and not in PLI- and PL2-crystal- lins.’5,20 PSI is also unusual in having a large number of hydrophobic residues on its surface.29 Our present results raise the question of whether, amongst the various mono- mers, PB 1 is particularly photovulnerable and its structure and association properties more readily perturbed. Our re- sults suggest that the dynamics of the quaternary structure of P-crystallins can be perturbed by environmental factors. We believe that this is of relevance to the in vivo situation

I i I I

h , n m A ,nm Figure 3. Near (A) and far (B) UV CD spectra of j3-crystallin after irradiation at 295 nm with various time spans: (-) 0 h, (- - -) 1 h, (-.-.-) 2 h and (. ’. . .) 4 h. Irradiation of crystallin solutions was done using the 150 W Xe lamp of the Hitachi F-4000 fluores- cence spectrophotometer with the monochromator at 295 nm and

the slit at 20 nm for various time spans.

0 5

A

400 SO0 600 700

A, nm

I I 580 620 660

V u -9

X,nm Figure 4. A Absorption spectra of the normal and irradiated j3-crys- tallin-stains-all complex. j3-crystallin was irradiated at 295 nm for 30 min and then incubated with dye. Dye : protein molar ratio was 3.9. (-) dye only; (. . . . .) dye + 0-crystallin; (---) dye + irra- diated j3-crystallin. B: Circular dichroism spectra of the normal (-) and irradiated (- - -) P-crystallin4ye complex with the above dye : protein molar ratio. The binding of the calcium-binding probe dye to the various crystallins was studied in 2 mMMOPS containing

30% ethylene glycol, pH 7.2, as described

such as in aging and cataract of the lens. Alcala et aL30 have seen that the composition of human P-crystallins displays specific changes with age and region of the lens. We are currently studying these aspects of the individual members of the P-crystallin family.

Efect of light on the calcium-binding ability of P-crystallin

We next studied the effect of photodamage on the calcium- binding ability of P-crystallin. The method chosen to assay the ion-binding ability was to use the calcium ion mimic dye Stains-all. This cationic carbocyanine dye displays specific absorption bands in the J region (620 nm) and/or the y region (500 nm) upon binding to the Caf2-binding regions of various calcium-binding p r ~ t e i n s - ~ l - ~ ~ Figure 4A shows that Stains- all binds to fresh, unirradiated solutions of both PH- and PL- crystallins and displays the J band in conformity with our earlier findingsz4 On the other hand, this diagnostic J band is not activated when the dye is incubated with P-crystallin that has been irradiated for 30 min at 295 nm. This differ- ential behavior is more dramatically seen in Fig. 4B, which shows that fresh P-crystallin binds to Stains-all well enough to be able to induce optical activity in the dye. P-crystallin, which has been irradiated even for 30 min at 295 nm, is not able to induce the J band of the dye both in absorption and CD. There is thus a substantial decrease in the calcium- binding affinity of P-crystallins upon irradiation.

These results might be relevant to the finding that the levels of free calcium in young, aged and cataractous lenses are different.z’,34 In young rat lenses, less than 1% of the total calcium is in the free form, while it is far higher in aged and

742 YOGENDRA SHARMA et al.

cataractous lenses. Cataract is reported both in cases of hy- pocalcemia and hypercalcemia. 0-crystallins constitute about 40% o f the total crystallins and can thus bind and sequester high amounts of calcium ions in the lens. Our results here show that the calcium-binding ability of P-crystallin is re- duced upon photomodification. Therefore, it appears that P-crystallin might help in controlling the levels of free cal- c ium associated with the opacity. Interestingly, Seccia et a[.-” reported that the susceptibility o f PL-crystallin t o calcium- mediated transglutaminase activity is augmented upon free radical oxidation. Photooxidation of Trp residue(s) in p-crys- tallin might subtly influence the structure of the calcium- binding site(s) or promote its ability to fold into an optimally active conformation. There are three Trp residues in the putative calcium-binding regions of various subunits of P - c r y ~ t a l l i n . ~ ~ It seems that some of these Trp might have a role in ion binding. With respect to ligand-binding properties of a protein, it is worth noting that even a small displacement of a liganding group could have a profound effect on binding affinity without affecting the secondary/tertiary structure sig- nificantly. We are currently investigating the calcium binding properties o f the individual members of the P-crystallin fam- ily.

Acknowledgements- We thank Ms. C. Subbalakshmi and V. Krishna Kumari for their help in performing the FFLC experiments. DB thanks the Jawaharlal Nehru Centre for Advanced Scientific Re- search, Bangalore, India, of which he is an honorary Professor.

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Research Note 743

that probes conformational features of calcium-binding pro- teins. In Novel Calcium-Binding Proteins- Fundamentals and Clinical Implications (Edited by C. Heizmann), pp. 5 1-6 1. Springer Verlag, Heidelberg.

34. Hightower, K. R. (1985) Cytotoxic effects of internal calcium on lens physiology: a review. Curr. Eye Res. 4, 453-459.

35. Seccia, M., D. Brossa, E. Gravela, T. F. Slater and K. H. Cheese- man ( 1 99 1 ) Exposure of#lL-crystallin to oxidizing free radicals enhances its susceptibility to transglutarninase activity. Bio- chem. J. 274, 869-873.