| Age-related cataract is a kind of disease which is closely related to the growth of age.A lot of factors in vitro and vivo contribute to the aging process or opacification of lens,such as free radical oxidative damage,long-term ultraviolet radiation,ionizing radiation and so on.In recent years,with the development of molecular biological technology,some in-depth studies of the crystallin proteome and post-translational modification have shown that age-related cataract belongs to a kind of disease with the structural changes of protein.In this study,we mainly discuss the relationship between the changes of proteins and enzymes in lens and age-related cataract.DPPⅢis a kind of exo-peptidase which is present in the cytoplasm of lens epithelial and lens fiber cells.At present,some scholars at home and abroad has revealed characteristics of DPPⅢby some related research:DPPⅢis a zinc metallo-exopeptidase whose active center is primarily serine.It has been identified and purified from cytosolic fractions of various mammalian tissues.The molecular weight of the purified enzyme was about 82kDa on SDS-PAGE.The cDNAstructure is composed of 2640 bp of nucleotides in length and encodes 738 amino acids in the coding region.And the enzyme has a novel zinc-binding motif,HEXXXH in structure.Until now seven sequences of DPPⅢfrom human Origin have been stored in databases.Human DPPⅢis a monomeric protein consisting of 737 amino Acids which is highly homologous (93.45%identity of the amino acids sequence)to its rat counterpart.It specifically hydrolyzes cleave dipeptides from the N-terminus of peptides and protein with an optimum pH of 7.5-9.5,such as Arg-Arg-, Ala- Arg-,Asp-Arg- or Tyr-Gry-.DPP-Ⅲmay be involved in the metabolism of very important bioactive Peptides such as enkephalins and angiotensinsⅡ,Ⅲ.Spinorphin(LVVYPWT) was found to be an endogenous factor modulates enkephalin-degrading enzymes.An inhibition assay involving various spinorphin analogues suggested that tynorphin(VVYPW) is the most potent inhibitor of DPPⅢ.A partial purification of dipeptidyl peptidaseⅢhas been achieved from human cataractous lenses.It was proved that the specific activity was increased 45.5 fold over that of the original aqueous extract.An immunohistochemical study on lenses derived from Shumiya cataract rats also suggested that the immunoreactivity of DPPⅢin lens fibres increased with age in cataract rats,but that of a B-crystallin decreased.Object:In order to find the role of DPPⅢ1 played during cataract formation,we choosed normal rat lenses as our study targets,and detect the expression of dipeptidyl peptidaseⅢin rat lenses of different weeks(because of the highly homology between human DPPⅢand rat DPPⅢ)Methods:Firstly,Western-blotting was used to examine the content of DPPⅢin rat lenses of different weeks.Then,enzyme activity of DPPⅢwas determined by directly monitoring the absorbrance with UV-2500PC at 525nm 37℃,according to the enzyme can removes the dipeptides from the special substrate(Arg-Arg- 4mbNA ).Results:The results of Western-blotting for examining the content of DPPⅢindicate that the content of DPPⅢin rat lenses increased with the growth of weeks.The results of enzyme activity of DPPⅢindicate that the enzyme activity of DPPⅢin rat lenses increased with the growth of weeks.Conclusion:Based on these results,the expression changes of DPPⅢin rats lenses with aging,maybe involved in the changes of crystallin at the process of lens development.For example:the content ofαB-crystallin dereased in soluble components but increased in insoluble components with aging,αB-crystallin has shown the feature of post-translational modification.It also be identified that it can be cleaved off from N-terminus.We conclude that DPPⅢplay an important role in hydrolyzing crystallin or modifying post-translation,in the process of lens aging or opacification.When the concentration of Ca2+ increased in lens,some endo-peptidases, such as calpains etc,are activated by Ca2+ in lens.So that some lens protein was firstly degraded into peptide fragments.The N-terminal specifical amino acid sequences of these polypeptide fragments,such as:Arg-Arg-,Ala- Arg- or Tyr-Gry- etc,are revealed,and then are recognized and hydrolyzed or post-translational modified by DPPⅢ.All these change the original structure and sequence of crystal protein or peptides.Thus,αA-,αB-crystallin becomes insoluble, giving rise to transparency changes of lens.It demonstrates that DPPⅢmay act an important function in the formation and the aggravation of age-related cataract. |
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