Page 2: Corrective Lenses

In Kaplan’s lab, Allan Wilson also helped characterize LDHs with respect to their amino acid composition, catalytic properties, and thermal stability (12, 13). Among the phylogenetic patterns observed was the unusual heat stability of the H₄ isozyme from most birds and some reptiles (13). Why should these LDHs be so stable (75C < T_m < 85C, where T_m is the denaturation temperature.)? Did this reflect a response to some selective pressure in those organisms? Or, was this an in vitro characteristic of no biological importance? An unexpected discovery puts these questions in perspective. The eyes have it (14).

The lens of the vertebrate eye is a terminally differentiated tissue with remarkable properties as noted in the introductory quotation by Darwin. The transparent cells of the lens have lost their nuclei and organelles. They contain large amounts of a few proteins called crystallins that must not denature in a lifetime. Crystallins must be transparent and stable. In addition to the generally distributed alpha, beta, and gamma-crystallins, there are crystallins restricted to certain taxa (15, 16). The lenses of most birds and crocodilians contain epsilon-crystallin, which can constitute up to 23% of the total lens protein. epslon-Crystallin is identical to LDH-H₄ (14, 17, 18)! Evolution is opportunistic, recruiting and adapting what is available. Apparently LDH-H₄ is but one of a number of enzymes that have been recruited as crystallins in various groups of organisms (15, 16, 31, 32).

Some learning issues to pursue.

• Thousands of enzymes exist. What properties of LDH and/or its structural gene might have predisposed its recruitment as a crystallin? In other words, what features might give LDH a selective advantage for being a lens protein over other proteins? [See ref. 19 for one not-so-apparent property.]
• epsilon-Crystallin occurs in birds and crocodilians whose most recent common ancestor lived more than 200 million years ago. It seems likely that this protein has been under dual selection to be simultaneously a good crystallin and a good LDH. Should selection for one function compromise selection for the other (20)? Is there observations one could make that might might suggest evolutionary constraints?
• While gene duplication (or genome duplication (22, 23)) generated the Ldh-A, -B, and -C loci that have evolved to have distinctive regulatory properties and encode proteins with distinctive kinetic properties, there is no evidence that the Ldh-B locus in birds has duplicated. Do you think selection would favor a duplication of the Ldh-B gene in some bird species (24)? Why? If duplication occurred, what evolutionary changes would you expect to occur in the resulting proteins?
• Among the avian heart-type LDHs studied by Wilson et al. (13) were several that were relatively heat sensitive (T_m < 70C). Suggest and discuss the basis for at least two testable evolutionary hypotheses might follow from this observation (21).
• The T_m values determined by Wilson et al. (13) were defined as the temperature at which LDH lost half of its activity in 20 minutes. For the sake of argument, assume that for a lens to function properly throughout a human life span of about 80 years, no more than 10% of its crystallin protein can denature. If the activation energy for protein denaturation is 50 to 100 kcal/mol (30), what should be a reasonable range of T_m values for a human crystallin?
• While much emphasis is placed on the selective pressures on protein function, there are selective effects on a gene that has been recruited to a new function. What changes in the regulatory control of an Ldh-B gene would be needed to express LDH as a crystallin in lens tissue (25, 26)?
• What would you predict about the rate of evolution of LDH-B in birds compared to mammals? Explain.
• The blind mole rat, Spalax ehrenbergi, lives its entire life in a maze of subterranean passages (27, 28). Its small vestigial eyes are beneath the skin. Its alphaA-crystallin gene has been cloned, sequenced, and compared to other mammalian alphaA-crystallins (29). Whereas alphaA-crystallins from rodent relatives such as mouse, rat, gerbil, hamster, and squirrel are identical to each other, that from the mole rat differs by nine amino acid replacements. A molecular phylogeny based on alphaA-crystallin would thus indicate that the mole rat is much less closely related to other rodents than it really is. Interpret these results in terms of various concepts in protein evolution.

References:

13. Wilson, A. C., Kaplan, N. O., Levine, L., Pesce, A., Reichlin, M. and Allison, W. S. (1964) "Evolution of lactate dehydrogenases" Fed. Proc. 23, 1258-1266.
14. Wistow, G. J., Mulders, J.W.N. and de Jong, W. W. (1987) "The enzyme lactate dehydrogenase as a structural protein in avian and crocodilian lenses" Nature 326, 622-624.
15. Wistow, G. and Piatigorsky, J. (1987) "Recruitment of enzymes as lens structural proteins" Science 236, 1554-1556
16. de Jong, W. W., Hendriks, W., Mulders, J.W.M. and Bloemendal, H. (1989) "Evolution of eye lens crystallins: The stress connection" TIBS 14, 365-368.
17. Hendriks, W., Mulders, J.W.M., Bibby, M. A., Slingsby, C., Bloemendal, H. and de Jong, W. W. (1988) "Duck lens epsilon-crystallin and lactate dehydrogenase B₄ are identical: A single-copy gene product with two distinct functions" Prof. Natl., Acad. Sci. USA 85, 7114-7118.
18. Kraft, H. J., Hendriks, W., de Jong, W. W., Lubsen, N. H. and Schoenmakers, J.G.G. (1993) "Duck lactate dehydrogenase B/sepsilon-crystallin gene--lens recruitment of a GC-promotor" J. Mol. Biol. 229, 849-859.
19. Zigler, J. S., Jr. and Rao, P. V. (1991) "Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens" FASEB J. 5, 223-225.
20. Wistow, G., Anderson, A. and Piatigorsky, J. (1990) "Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses" Proc. Natl. Acad. Sci. USA 87, 6277-6280.
21. Voorter, C.E.M., Wintjes, L.T.M., Heinstra, P.W.H., Bloemendal, H. and de Jong, W. W. (1993) "Comparison of stability properties of lactate dehydrogenase B₄/epsilon-crystallin from different species" Eur. J. Biochem. 211, 643-648.
22. Ohno, S. (1973) "Ancient linkage groups and frozen accidents" Nature 244, 259-262.
23. Fisher, S. E., Shaklee, J. B., Ferris, S. D. and Whitt, G. S. (1980) "Evolution of five multilocus isozyme systems in the chordates" Genetica 52/53, 73-85.
24. Piatigorsky, J. and Wistow, G. (1991) "The recruitment of crystallins: New functions precede gene duplication" Science252, 1078-1079.
25. Piatigorsky, J. (1989) "Lens crystallins and their genes: Diversity and tissue-specific expression" FASEB J. 3, 1933-1940.
26. Piatigorsky, J. (1992) "Lens crystallins--innovation associated with changes in gene regulation" J. Biol. Chem. 267, 4277-4280.
27. Honeycutt, R. L. (1992) "Naked mole-rats" American Scientist 80, 43-53 (describes the biology of relatives of the blind mole rats).
28. Kass, J. H. (1993) "Vision in blind mole rats" Nature 361, 113.
29. Hendriks, W., Leunissen, J., Nevo, E., Bloemendal, H. and de Jong, W. W. (1987) "The lens protein alphaA-crystallin of the blind mole rat, Spalax ehrenbergi: Evolutionary change and functional constraint" Proc. Natl. Acad. Sci. USA 84, 5320-5324.
30. Creighton, T. E. (1990) in Protein-Folding - Deciphering the Second Half of the Genetic Code. (Gierasch, L. M. & King, J., Eds.) Pp 157-170, AAAS, Washington, DC.
31. Tomarev, S. I. and Piatigorsky, J. (1996) Lens crystalins of invertebrates: Diversity and recruitment from detoxification enzymes and novel proteins. Eur. J. Biochem. 235, 449 - 465.
32. Riddihough, G. (1994) One in the eye Nature 371, 538.