The Transsulfuration Pathway in the Lens

Abstract

Abstract: : Purpose: The finding that a lens under oxidative stress accumulated free and protein-bound (protein-S-S-cysteine) cysteine in the fiber cells prompted us to examine if there is an alternative source for cysteine pool besides the active cysteine transport system in the lens, namely, the transsulfuration pathway of homocysteine-cystathionine-cysteine, that utilizes methionine through transmethylation. Methods: The presence of the gene for cystathionine-beta-synthase (CBS), the rate limiting enzyme that converts homocysteine to cystathionine, in human lens epithelial cells was studied by PCR method using primers designed based on the sequence of human liver CBS (Forward- 5’-CCA CAC TGC CCC GGC AAA AT- 3,’ Reverse--5’-CTG GCA ATG CCC GTG ATG GT-3.’).The PCR product was purified using QIA quick gel extraction kit. The presence of CBS enzyme in HLE cells or the distribution in the pig or human lens was verified using western blot analysis using human liver anti-CBS antibody (gift from R. Banerjee of University of Nebraska). The catalytic activity was assayed by measuring the production of C¹⁴-cystathionine from C¹⁴-serine in the presence of homocysteine, adanosine-methionine and xxxx. Results: The purified DNA fragment (580 bp) from PCR analysis was sequenced and confirms the homology with CBS gene from other human tissues. The presence of CBS protein band (67kDa) in the HLE cells reacted positively with the human liver anti-CBS antibody. This enzyme was found in the pig and human lens with highest intensity in the epithelial layer and lower but equal in quantity in the cortical and nuclear region. CBS activity was detectible in the human lens epithelial B3 cells. H2O2 (0.1 mM) treatment upregulated the mRNA and gene product of CBS. Same phenomenon was also observed when pig lens cultured under 0.5 mM H2O2. Conclusion: This is a first evidence that a transsulfuration pathway is present in the lens which may be upregulated under oxidative stress and provide more redox potential to the cells. Supported by NIH grant EY 10590. None

Keywords: metabolism • enzymes/enzyme inhibitors • oxidation/oxidative or free radical damage