Lens Crystallin Proteins from 3.28 Million Year Old Crocodilian, the Red Caiman (Caiman yacare): Yields Insights Into Protein Evolution and Structure

The global population is aging, and as a consequence, the number of individuals experiencing age-related symptoms is increasing. Senile cataracts occur when the eye lens becomes cloudy due to the buildup of unfolded, or denatured, proteins over time. Cataracts are the leading cause of blindness worldwide and cause a significant decline in quality of life. They are particularly difficult for individuals who live in societies that have minimal accommodations for the sight-impaired. To better understand the formation and structure of cataracts, and the lack thereof in animals that are resistant to cataract formation, we isolated and sequenced the lens crystallin cDNAs from the red caiman. Lens crystallins are the proteins in the eye lens that impart the transparent optical nature to this tissue, and when these crystallins are disrupted, the result is cataract formation. Caimans, being crocodilian, are an evolutionarily ancient and very successful group of animals, meaning that their genome is well-adapted to life on earth. Being cold-blooded, aquatic, and native to the tropics, caimans are subject to a lot of environmental stress. This makes them an ideal candidate to study regarding life-long lens crystallin structure and stability. Lens crystallins, like many proteins, come in a variety of subtle variations – called isoforms. These isoforms arise due to small changes in the genetic code that cause changes in key amino acids during alternative splicing. These variants all have similar, but slightly different structures when folded into their final forms as proteins. We used a novel technology that uses full-length isoform sequencing (Iso-Seq), a method of long-read cDNA sequencing, to inventory the mRNA transcript-level variations in the caiman lens crystallins that result in their many isoforms. With this data, we hope to pinpoint not only the isoforms that may be particularly stable or resistant to stress, but also any mutations at specific amino acid residues that make them uniquely resistant to senile insult. Current treatment for cataracts is surgical, but with the increasing aging population, cataract surgery may prove to be ineffective at meeting the demand for cataract treatment. Therefore, the hope is for this data to be used in the development of a non-surgical treatment for cataracts, such as a prophylactic treatment similar to fluorination of drinking water, or the addition of folic acid to cereal grains.

Our work centered around isoform analysis to identify those that are particularly stable or resistant to stress, along with the mutations at specific amino acid residues responsible for their characteristic structural integrity. Because lens crystallins are highly conserved, we opted to use a novel technology for our isoform analysis. The PacBio Vega was recently acquired by the Center for Bioinformatics and Functional Genomics (CBFG) here at Miami. We used the PacBio Vega to perform our isoform analysis, which uses full-length isoform sequencing (Iso-Seq), a method of long-read cDNA sequencing, to inventory the mRNA transcript-level variations in the caiman lens crystallins that result in their many isoforms. Previously, this type of analysis would have had to have been done by short-read cDNA sequencing, which essentially chops up and compares pieces of each isoform sequence, then puts them back together. However, the short fragments can be hard to keep track of, especially for highly conserved proteins like lens crystallins, because they have so many domains with the same sequence. Long-read sequencing allowed us to have a more streamlined analysis by removing the step of rebuilding the whole isoform sequence, which also reduces error.

Next steps for this project involve continuing to sort through our sequence data to further narrow down which sequences are legitimate isoforms. Beyond this, we could use mass spectrometry to validate which isoforms are expressed (as protein) in the lens, since not all mRNAs end up being expressed as proteins. We could also use in silico structural models for the crystallins to better determine residue placement, and therefore function, in stability. Finally, we could also correlate isoform expression with epigenetic markers in lens DNA by sequencing the Red Caiman genome and mapping the isoforms back to it.

Regarding clinical applications, this data could aid in the development of a non-surgical treatment for cataracts, such as a prophylactic, like folic acid in breakfast cereal. A prophylactic treatment would be more accessible than the current standard treatment for cataracts, cataract removal surgery. This may be able to better meet the rising demand for cataract treatments that is occurring in tandem with the aging global population.

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