Protein Modifications and Lipid Composition Changes in Rat Lenses in Postnatal Development

The aim of the investigation is to study age dynamics of posttranslational protein modification level and the changes of rat lens membranes, and the consideration of possible mechanisms of membrane effect on the composition and intensity of protein modifications in lens.

Materials and Methods. The experiments were carried out on Wistar rats of three age groups: 1, 12 and 24 months. Protein level, sulfhydryl (SH) group concentration, and protein carbonyl derivatives level were measured spectrophotometrically. The content of tryptophan, bityrosine and advanced glycation end-products (AGEs) were assessed by fluorescence intensity. Phospholipids and neutral lipids were fractionated by thin-layer chromatography. Densitometric analysis and quantitative processing of chromatograms were performed using NIH Image J software.

Results. Protein content in lens homogenate was found to increase with age, indicating the accumulation of slightly soluble protein aggregates. There was uniform decrease of SH-group concentration and protein carbonyl derivatives in homogenate. On the other hand, there was observed the accumulation of AGEs, bityrosine and tryptophan in water-soluble fraction. The main age changes of lens membrane lipid composition were the increasing ratio of sphingomyelin and neutral lipids. The changes could be caused by the growth of the proportion of mature fibers forming the nucleus of lens compared to poorly- and medium-moderately fibers and cells of epithelium. The principal component of neutral lipids was cholesterol and cholesterol esters.

Conclusion. Lens membrane enrichment by lipids characterized by relatively high “ordering” inhibits the formation of protein carbonyl derivatives, but at the same time, can disbalance intercellular communication resulting in proteolysis (and tryptophan exposure) and AGEs accumulation.

1. Bassnett S. Fiber cell denucleation in the primate lens. Invest Ophthalmol Vis Sci 1997; 38: 1678–1687.
2. Sakthivel M., Elanchezhian R., Thomas P.A., Geraldine P. Alterations in lenticular proteins during ageing and seleniteinduced cataractogenesis in Wistar rats. Mol Vis 2010; 16: 445–453.
3. Hanson S.R., Hasan A., Smith D.L., Smith J.B. The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage. Exp Eye Res 2000; 71: 195–207.
4. Kopylova L.V., Cherepanov I.V., Snytnikova O.A., Rumyantseva Y.V., Kolosova N.G., Tsentalovich Y.P., Sagdeev R.Z. Age-related changes in the water-soluble lens protein composition of Wistar and accelerated-senescence OXYS rats. Mol Vis 2011; 17: 1457–1467.
5. Friedrich M.G., Truscott R.J.W. Membrane association of proteins in the aging human lens: profound changes takes place in the fifth decade of life. Invest Ophthalmol Vis Sci 2009; 50: 4786–4793.
6. Yappert M.C., Rujoi M., Borchman D., Vorobyov I., Estrada R. Glycero-versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth. Exp Eye Res 2003; 76: 725–734.
7. Borchman D., Yappert M.C. Lipids and the ocular lens. J Lip Res 2010; 51: 2473–2488.
8. Truscott R.J.W. Age-related nuclear cataract-oxidation is the key. Exp Eye Res 2005; 80: 709–725.
9. Raju I., Kumarasamy A., Abraham C.E. Multiple aggregates and aggresomes of C-terminal truncated human αA-crystallins in mammalian cells and protection by βB-crystallin. PLoS ONE 2011; 6(5): e19876. doi:10.1371/journal.pone.0019876.
10. Luthra M., Balasubramanian D. Nonenzymatic glycation alters protein structure and stability. A study of two eye lens crystallins. J Biol Chem 1993; 268: 18119–18227.
11. Riddles P.W., Blakeley R.L. Zerner B. Reassessment of Ellman’s reagent. Methods Enzymol 1983; 91: 49–60.
12. Dubinina E.E., Burmistrov S.O., Hodov D.A., Porotov I.G. Voprosy meditsinskoy khimii — Medical Chemistry Issues 1995; 1(41): 24–26.
13. Muthenna P., Akileshwari C., Saraswat M., Reddy G.B. Inhibition of advanced glycation end-product formation on eye lens protein by rutin. Br J Nutr 2011. doi:10.1017/S0007114511004077.
14. Folch P.J., Lees M., Stanley G. A simple method for the isolation and purification of total lipids from animal tissue. J Biol Chem 1957; 226: 497–509.
15. Sharshunova M., Shvarc V., Mihalec Ch. Tonkosloynaya khromatografiya v farmatsii i klinicheskoy biokhimii. Ch. 2 [Thin layer chromatography in pharmacy and clinical biochemistry. Part 2]. Moscow: Mir; 1980; p. 536–540.
16. Khromatografiya. Prakticheskoe prilozhenie metoda Ch. 1. [Chromatography. Practical application of the technique. Part 1]. Pod red. Kheftmana E. [Kheftman E. (editor)]. Moscow: Mir; 1986; 336 p.
17. Glants C. Mediko-biologicheskaya statistika [Biomedical statistics]. Moscow: Praktika, 1998; 459 p.
18. Santhoshkumar P., Murugesan R., Sharma K.K. αA-Crystallin peptide 66SDRDKFVIFLDVKHF80 accumulating in aging lens impairs the function of α-crystallin and induces lens protein aggregation. PloS ONE 2011; 6(4): e19291. doi:10.1371/journal.pone.0019291.
19. Nemet I., Monnier V.M. Vitamin C degradation products and pathways in the human lens. J Biol Chem 2011; 286(43): 37128–37136.
20. Fan X., Zhang J., Theves M., Strauch C., Nemet I., Liu X., Qian J., Giblin F. J., Monnier V.M. Mechanism of lysine oxidation in human lens crystallins during aging and in diabetes. J Biol Chem 2009; 284: 34618–34627.
21. Sell D.R., Monnier V.M. Ornithine is a novel amino acid and a marker of arginine damage by oxoaldehydes in senescent proteins. Ann NY Acad Sci 2005; 1043: 118–128.
22. Raguz M., Widomska J., Dillon J., Gaillard E.R., Subczynski W.K. Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches. Biochim Biophys Acta 2008; 1778: 1079–1090.
23. Oborina E.M., Yappert M.C. Effect of sphingomyelin versus dipalmitoyl-phosphatidylcholine on the extent of lipid oxidation. Chem Phys Lipids 2003; 123: 223–232.
24. Morales A., Lee H., Goni F., et al. Sphingolipids and cell death. Apoptosis 2007; 12: 923–939.
25. Tong J., Briggs M.M., Mlaver D., Vidal A., McIntosh T.J. Sorting of Lens Aquaporins and Connexins into Raft and Nonraft Bilayers: Role of Protein Homo-Oligomerization. Biophys J 2009: 97: 2493–2502.
26. Liu J., Xu J., Gu S., Nicholson B.J., Jiang J.X. Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50. J Cell Sci 2011; 124: 198–206.
27. Mathias R.T., White T.W., Gong X. Lens gap junctions in growth, differentiation and homeostasis. Physiol Rev 2010; V90: 179–206.
28. Biju P.G., Rooban B.N., Lija Y., Gayathri D.V., Sahasranamam V., Abraham А. Drevogenin D prevents selenite-induced oxidative stress and calpain activation in cultured rat lens. Mol Vis 2007; 13: 1121–1129.
29. Yappert M.C., Borchman D. Sphingolipids in human lens membranes: an update on their composition and possible biological implications. Chem Phys Lipids 2004; 129: 1–20.
30. Grami V., Marrero Y., Huang L., Tang D., Yappert M.C., Borchman D. α-Crystallin binding in vitro to clear human lenses. Exp Eye Res 2005; 81: 138–146.

Knyazev D.I., Ivanova I.P. Protein Modifications and Lipid Composition Changes in Rat Lenses in Postnatal Development. Sovremennye tehnologii v medicine 2012; (4): 36