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First published online December 3, 2004
Journal of Experimental Biology 207, 4633-4649 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01312

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Cold-stable eye lens crystallins of the Antarctic nototheniid toothfish Dissostichus mawsoni Norman

Andor J. Kiss¹, Amir Y. Mirarefi², Subramanian Ramakrishnan³, Charles F. Zukoski^2,3, Arthur L. DeVries¹ and Chi-Hing C. Cheng^1,*

¹ Department of Animal Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA
² Centre for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA
³ Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA

View larger version (59K): [in a new window]   Fig. 1. Lens shapes and cold-cataract cooling experiment on the lenses of three species (Bos taurus, Myripristis jacobus and Dissostichus mawsoni) from three different physiological temperatures (37°C, 25°C and –2°C, respectively). (a–d) Schematics of the shapes (a,c) and a picture (b,d) of a D. mawsoni (a,b) and B. taurus (c,d) lens. (e,f) Cold-cataract experiment results showing lenses from the cow, B. taurus. (e) A fresh lens at room temperature (25°C); (f) a bovine lens that had been packed in ice for approximately 1.5 h, from which it was removed and allowed to warm. This image was taken during the warming process after the cortex had rapidly clarified and the nucleus was still opaque, showing the cold-cataract (arrow). (g–i) The eye lens from the tropical marine blackbar soldierfish M. jacobus held at (g) 15°C for 6 h; (h) 0°C for 6 h; (i) 0°C for 48 h, showing a definite inner nuclear region that is more opaque that the cortex region (arrow). (j–k) Images of the Antarctic toothfish D. mawsoni eye lens. (j) Endogenous clear –2°C lens contrasted to a lens held at –12°C for 6 h (k). The still clear toothfish lens after 48 h at –12°C (l) has a thin sheen of opacity. It is important to note that the opacity is restricted to the surface and not to the inner portions of the lens, as in the cow (f) and the soldierfish (i). Scale bars, 1.2 cm (e), 0.4 cm (g), 1.0 cm (j).

View larger version (16K): [in a new window]   Fig. 2. Size-exclusion chromatography using Sephacryl 200 High Resolution resin of crystallins from three species, D. mawsoni (A), T. obesus (B) and B. taurus (C). Peaks are labelled as /ßH (peak I), ßH (peak IIa), ßL (peaks II or IIb), or S and (peaks IIIa and III). Numbers labelled on each individual peak correspond to the lanes of the SDS-PAGE and immunoblots presented in Fig. 3. Fraction volume sizes are 5 ml each.

View larger version (72K): [in a new window]   Fig. 3. SDS-PAGE (A,E,I) and immunoblot analysis (B–D, F–H, J–L) of SEC elution profiles of crystallins from D. mawsoni (A–D), T. obesus (E–H) and B. taurus (I–L). The primary antibodies used for detection are denoted on the left. Molecular mass markers (Lane M; kDa) are denoted on the left of each panel. Lane numbers correspond to the locations of the numbered positions on Fig. 2. for each species. All immunoblots are independent replicas of SDS-PAGE presented in Fig. 3A,E,I.

View larger version (19K): [in a new window]   Fig. 4. Determination of the upper limit of thermal stability TS for the (A) and (B) crystallins from D. mawsoni, measured as the change in turbidity (A360) over time at various incubation temperatures. Concentration of crystallins was 1 mg ml–1 in all assays. (A) Upper limit of TS for crystallin. At temperatures above 47°C, turbidity increases with time. at 47°C (x); 55°C (x); 60°C (x). (B) Upper limit of TS for crystallin. at 30°C (x); 33°C (x); 35°C(x); 42.5°C (x); 44°C (x); 45°C (x); 47°C (x); 57.5°C (x). The TS was determined as 47°C for the and 33°C for the crystallin.

View larger version (35K): [in a new window]   Fig. 5. Chaperone protection by crystallin of heat-aggregated crystallin (A–C) along with protection of DTT-denatured lysozyme (D–F). (A,D) D. mawsoni; (B,E) T. obesus; (C,F) B. taurus. Temperatures for the assay are indicated in each panel. Values are means ± S.E.M. (N=3). In A–C the crystallin and crystallin are both from the same species, with fractional amounts of crystallin added based on a 1:1 mass ratio of to crystallin at a final assay concentration of 1 mg ml–1 for both crystallins (i.e. x in A–C). In D–F the amount of crystallin added was based on a 1:1 mass ratio of crystallin to lysozyme, where in all assays (D–F) there was 0.2 mg ml–1 of lysozyme containing 20 mmol l–1 DTT. For A–C: x ; x 1:16 :; x 1:8 :; x 1:4 :; x 1:2 :; x :. For D–F: x 0:1 :lyso; x 1:1 :lyso; x 2:1 :lyso; x 3:1 :lyso; x 2.5:1 :lyso; x 5:1 :lyso; x 7.5:1 :lyso; x 10:1 :lyso; x 11:1 :lyso.

View larger version (19K): [in a new window]   Fig. 6. Cross-species chaperone protection assay of crystallin by crystallin from the three species D. mawsoni, T. obesus and B. taurus. Chaperone assay temperature was at TS for the crystallin in the assay. Final concentration of both and crystallin in the assay was 1 mg ml–1. Combinations of and crystallin are indicated. Values are means ± S.E.M.; bars are obscured by symbols (N=3).

View larger version (13K): [in a new window]   Fig. 7. Functional assessment of B. taurus crystallin after 1 h incubation at 60°C in the presence of D. mawsoni crystallin. (A) Experimental mg B. taurus crystallin + 1 mg D. mawsoni crystallin (x) and control reaction of mg B. taurus crystallin + 1 mg B. taurus crystallin (x). After 1 hsamples were removed from the cuvette and any precipitate was removed by centrifugation at 21 000 g at 4°C in a benchtop microcentrifuge. (B) Supernatants (750 µl) were then transferred to a new cuvette and a further 1 mg of B. taurus crystallin was added. B. taurus crystallin was concentrated such that the total volume in the second assay (after addition of the B. taurus crystallin) was not greater than 1 ml. Final composition of assay was (B. taurus + D. mawsoni ) supernatant + B. taurus crystallin (x) for the experimental and (B. taurus + B. taurus ) supernatant + B. taurus crystallin (x). Second assay incubation was for 1 h at 60°C.