For the first time, RIDC Redoxoma researchers led by Professors Ohara Augusto and Graziella Ronsein, from the Instituto de Química at Universidade de São Paulo (USP), demonstrated the presence of tryptophan and tyrosine cross-links in human cataract samples, produced by radical mechanisms. The formation of cross-links and the consequent aggregation of proteins are modifications, oxidative or not, which are the main factors that contribute to lens opacity.

“The key to our work is that this is the first time that two types of cross-links, tryptophan-tyrosine and tryptophan-tryptophan, were unequivocally identified by mass spectrometry in human samples that are extremely complex, containing several protein modifications. At this point, the relevance of this work is more scientific than clinical. If these cross-links are involved in the pathogenic mechanism of cataract will have to be studied. We are opening perspectives for new studies. Because it is a radical modification, some antioxidants such as nitroxides, glutathione, or ascorbic acid can be effective in stopping the process. This is a possibility to be tested. It would be very important to be able to prevent or delay the development of cataracts”, Ohara Augusto said.

Like the lens of a camera, the lens in our eyes has the function of focusing the light on the retina for a clear view, in addition to adjusting the focus of the eye, allowing us to see clearly near and far. A cataract is the clouding of the lens. It is the most common cause of vision loss in people over 40 and the leading cause of blindness in the world. According to the World Health Organization (WHO) first world report on vision published in 2019, there are more than 65 million people with cataract in the world. As the population ages, this number tends to increase. Currently, the only treatment available is surgical removal of the lens and implantation of artificial intraocular lenses. This procedure is generally effective, but not without the risk of complications.

“Cataract is a disease that affects millions and there is still no definite mechanism on how it develops. It is known that protein aggregation promotes structural destabilization of crystallins leading to the opacification of the lens. Our work contributes to the understanding of this process to make possible cataract treatment in the future”, said Verônica Paviani, first author of the article published in the journal Free Radical Biology & Medicine (FRBM).

The study was carried out during Paviani’s doctorate – currently, she is a postdoctoral fellow at the Feinberg School of Medicine of Northwestern University, in Chicago – with the collaboration of Dr. Amaryllis Avakian’s group, head of the Cataract Surgery Department of the Hospital das Clínicas, FMUSP, and Professor Paolo Di Mascio, from the IQ-USP and a member of the RIDC Redoxoma.

Sunlight

The human lens is composed largely of crystallin proteins (alpha, beta, and gamma-crystalline) assembled in a highly ordered and interactive macro-structure essential for the transparency of the lens and the index of light refraction. These proteins do not exhibit turnover, that is, they are not degraded and replaced, remaining the same throughout our lives. Consequently, they are subject to various post-translational modifications, which are changes that proteins undergo after synthesis.

Another characteristic of these proteins is a large number of tryptophan residues, which are easily oxidized, in addition to being the ones that most absorb light in the ultraviolet (UV) region, among the 20 amino acids that make up proteins. The oxidation of tryptophan leads to the formation of the tryptophanyl radical, which recombines producing a cross-link and dimer. Protein cross-links refer to the formation of covalent bonds between two side chains of amino acids. According to the researchers, although it has been known since the 1940s that tryptophan is sensitive to oxidation by light, no one had detected this dimerization. In 2010, Ohara’s group first characterized the formation of a ditryptophan cross-link in the human superoxide dismutase enzyme (hSOD1) during its bicarbonate-dependent peroxidase activity, in this case by carbonate radical-mediated reactions.

“In my doctorate, we found that UV light also promotes the formation of tryptophan ross-links, and that gave us the idea of looking for this kind of modification in cataract, which is a context that involves oxidation by light”, Verônica said. There is much evidence that the continuous light to which we are exposed from birth has a role in the development of cataracts.

To investigate this hypothesis, the researchers analyzed the lens proteins of three patients aged 64 to 78 years with nuclear cataracts in an advanced stage. Using the technique of Liquid Chromatography Coupled to Mass Spectrometry (LC-MS / MS), they investigated and characterized the cross-links tryptophan-tryptophan, tryptophan-tyrosine, and tyrosine-tyrosine in the samples. The lenses were collected from patients diagnosed and operated on by Drs. Paulo J de Melo and Amaryllis Avakian. In parallel experiments, they irradiated the bovine beta crystallins with a solar simulator and verified the formation of similar cross-links. The bovine beta crystallins were chosen because they have great homology with the human ones, that is, the protein sequences are very similar.

The identification of these changes is not trivial. “These aggregates can be easily visualized by an SDS-PAGE experiment, in which we see the protein bands on a gel. However, this simple methodology does not allow identification either the proteins or the amino acids involved in the cross-link. We employed mass spectrometry, a very accurate methodology that resolves the above problems”, Verônica explained.

The challenge of mass spectrometry here was to analyze thousands of overlapping peptide spectra to identify the modifications of interest. “What we did was to develop a method to select the peptides that were forming cross-links. Today there is software to look for protein modifications, but, so far, we do not have software that are good enough to identify these types of cross-links. That is why the challenge was so great”, she added.

“We used very strict criteria to search for cross-links and probably because of that we characterized a small number of them but in a very consistent way. I believe that many other of these cross-links must be present in cataracts”, Ohara concluded.

The article Human cataractous lenses contain cross-links produced by crystallin-derived tryptophanyl and tyrosyl radicals, by Verônica Paviani, Paulo Junqueira de Melo, Amaryllis Avakian, Paolo Di Mascio, Graziella Eliza Ronsein and Ohara Augusto, can be accessed at https://www.sciencedirect.com/science/article/abs/pii/S0891584920312156