title: Effect of homocysteinylation on structure, chaperone activity and fibrillation propensity of lens alpha-crystallin.
abstract: Various chemical modifications can reduce chaperone activity of α-crystallin (α-Cry) and the loss of which has been implicated in the development of cataract diseases. The side chains of lysine residues are the target of both glycation and homocysteinylation, and lysine modification by the two reactions may similarly affect the structure and function of α- Cry. In this study, α-Cry was incubated with homocysteine thiolactone (HCTL), resulting in significant protein homocysteinylation, as determined with Ellman's assay. Homocysteinylation of α-Cry resulted in the reduction in surface hydrophobicity and alpha-helix to beta-sheet transition, as observed respectively with fluorescence and circular dichroism (CD) spectroscopy. The structural alteration of homocysteinylated α-Cry was accompanied by protein aggregation, including the formation of amyloid fibrils as detected by thioflavin T (ThT) fluorescence and Congo red (CR) absorption spectroscopy. The mobility shifts of homocysteinylated α-Cry on reducing and non-reducing SDS-PAGEs suggest that disulfide cross-linking in addition to lysine modification, also plays a role in aggregation of this protein. The chaperone activities of α-Cry, namely to prevent aggregation, to assist refolding and to restore activity of thermally stressed α-glucosidase (α-Gls) were reduced after homocysteinylation. Overall, this study suggests that similar to non-enzymatic glycation, homocysteinylation of α-Cry is a risk factor for the development of cataract disorders, for instance during hyperhomocysteinemia which is linked to the various ocular pathological disorders.
authors: Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran. ryousefi@shirazu.ac.ir
authors: Reza
authors: R
authors: Yousefi
authors: Sima
authors: S
authors: Khazaei
authors: Ali-Akbar
authors: AA
authors: Moosavi-Movahedi
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D002386:Cataract / Q000209:etiology / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D005123:Eye / Q000378:metabolism; D006031:Glycosylation; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D038201:alpha-Crystallins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000682:Amyloid; D038201:alpha-Crystallins; D006710:Homocysteine; C007957:homocysteine thiolactone