The presence of a transsulfuration pathway in the lens: a new oxidative stress defense system

Abstract

The finding that a lens under oxidative stress accumulated free and protein-bound cysteine (protein-S–S-cysteine) in the fiber cells prompted us to examine if there is an alternative source for cysteine pools besides the active cysteine transport system in the lens, namely, the transsulfuration pathway of homocysteine–cystathionine–cysteine, which utilises methionine through transmethylation. We examined the presence of the gene for cystathionine-beta-synthase (CBS), the rate limiting enzyme that converts homocysteine to cystathionine in the transsulfuration pathway, in human lens epithelial (HLE) B3 cells using PCR with 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 purified DNA fragment (586 bp) from PCR analysis was sequenced and confirmed the homology with CBS gene from other human tissues. The CBS protein band (67 kDa) was present in the HLE cells, which reacted positively with the human liver anti-CBS antibody. The enzyme protein was detected in the pig and human lenses with the highest intensity in the epithelial layer, lower but equal quantities of CBS was present in the cortical and nuclear regions. Human nuclear CBS increased while epithelial CBS decreased with aging. Oxidative stress transiently upregulated the gene expression of CBS both in HLE cells (0·1 m mH 2O 2) and in pig lens cultured in TC 199 medium (0·5 m mH 2O 2). The catalytic activity for CBS, which was assayed by measuring the production of C 14-cystathionine from C 14-serine in the presence of homocysteine, S-adenosyl-methionine and pyridoxal phosphate, was detectable in the HLE cells and transiently activated with H 2O 2. Free cystathionine accumulated when HLE B3 cells were treated with propargylglycine (PGG), an inhibitor of cystathionase, the downstream enzyme that converts cystathionine to cysteine. More cystathionine accumulation occurred when the cells were simultaneously exposed to PGG and 0·1 m mH 2O 2. We have shown that oxidative stress of H 2O 2 could increase the flux of this transsulfuration pathway by committing more homocysteine to cysteine and glutathione production as H 2O 2 (0·1 m m) inhibited the remethylation enzyme of methionine synthase while concurrently activating the CBS enzyme. This is the first evidence that a transsulfuration pathway is present in the lens, and that it can be upregulated under oxidative stress to provide additional redox potential for the cells.