Alpha Crystallin Evolution and Function
Alpha crystallin proteins help prevent the development of cataract during lens aging and appear to play a role in many other diseases from Alzheimer’s and multiple sclerosis to various cancers. The connection between alpha crystallin’s role in these diseases is its ability to bind to and prevent damage in other proteins. This protective chaperone activity changes with temperature, so we hypothesized that since fish body temperatures match their surrounding environment, unlike the constant high body temperatures in mammals, we could use fishes from diverse habitats to detail how alpha crystallin chaperone activity works.

We began by cloning the alpha crystallins from a tropical species called the zebrafish, which has become the “mouse” of fish research because they are easy to breed in captivity. After making the interesting discovery that fish like the zebrafish contain one more alpha crystallin than mammals, and characterizing these three proteins in a series of papers, we focused on one alpha crystallin in particular (the A version) and cloned that gene from five other fish species. We used these six fish alpha A-crystallins from species like the cold-bodied Antarctic toothfish and the temperate Ohio blunt-nosed minnow to figure out that each fish’s version had evolved to work at that species’ particular body temperature. The cold-adapted alpha A-crystallins are more flexible than the warm-adapted ones if you compare them at the same temperature, so at their normally frigid surroundings they are loose enough to interact with and protect damaged proteins.

Using computer software we were able to compare the shapes of the six fish alpha A-crystallins and predict specific amino acid changes that were responsible for the evolution of the protein’s function. We can test our predictions by engineering fish alpha A-crystallins with these changes and measuring their flexibility and protective chaperone function. The image above shows three amino acids mapped on a 3-dimensional computer drawing of alpha A-crystallin. Our latest experiments showed that one amino acid change in particular (the change of the valine at position 62 to threonine) significantly loosened alpha A-crystallin structure and made it a stronger chaperone. This work was recently presented by two undergraduate researchers from our lab at the annual meeting of the Association for Research in Vision and Opthalmology.

You can read more about the evolution of lens crystallins on my blog.

Role of Crystallins in Zebrafish Development
We are using a variety of techniques to investigate the possible importance of the three alpha crystallins in the development of the zebrafish embryo. The zebrafish has become a powerful model species for studying vertebrate development because females lay transparent eggs, allowing direct examination of the embryo in real time as it grows. In one set of experiments we are cloning the promoter regions for zebrafish alpha crystallins, the portions of DNA that determine where each gene is used to make protein. By connecting a gene for green fluorescent protein (GFP) to each promoter we can visualize where and when that promoter is active. We can also test the promoter regions from other species. The images above show a 2-day old zebrafish embryo under brightfield microscopy (A) and under fluorescent microscopy (B) indicating that the promoter region for mouse alpha A-crystallin can drive the expression of the attached GFP in the lens. We are using similar techniques to detail the function of the zebrafish promoters.

By using a synthetic anti-sense RNA called a morpholino we can also silence the production of individual alpha crystallin proteins in zebrafish embryos. We inject small quantities of each morpholino into zebrafish embryos when they contain only one to four cells (seen at the right with a red tracer dye in an injected embryo) using a picoinjector. The growing embryos can then be examined to see if the lack of specific alpha crystallins cause developmental abnormalities, suggesting possible roles for each.

Effect of Aging on the Zebrafish Lens
One of the fascinating things about the eye lens is its inability to make new proteins in most of its cells. The average cell in your body is constantly replacing proteins as they age, partially unfold, and lose their normal function. But to become transparent the inner cells of the lens, called fiber cells, lose their nuclei, and with them, the DNA blueprints they need to make new lens proteins. One of alpha crystallin’s functions is to bind to these aging proteins, preventing them from sticking together to produce cloudy masses, or cataract, in the lens.

We are using the zebrafish lens as a model system to examine what happens to proteins as the lens ages. In an initial paper we showed that zebrafish lens proteins age in a similar fashion to those in mammals, suggesting that we can use the zebrafish to ask questions about vertebrate lens aging and disease. One technique we use to detail the protein contents of the lens is 2D-gel electrophoresis, which allows us to separate a complex mixture of lens proteins into individual spots that each contain only one to several proteins. The protein in these spots can then be identified by mass spectrometry. The image above shows a typical set of protein spots from an adult zebrafish lens, with some major crystallin types labeled. We are now examining lens protein content from different ages to determine how this pattern changes during development.