Reversible Regulation of Human Lens Low Molecular Weight Protein Tyrosine Phosphatase by Oxidation

Abstract

Abstract: : Purpose: This study is to demonstrate the cloning and expression of human lens low molecular weight protein tyrosine phosphatase (LMWPTP) in human lens epithelial cells (HLE B3), and its reversible inactivation by oxidation. Methods: RT–PCR was applied to amplify LMWPTP cDNA from total RNA with primers designed based on the sequence of acid phosphatase from human red blood cells. PCR product was purified and cloned into pET23a (+) vector for sequencing. LMWPTP with His tag was overexpressed in E coli, purified, and characterized with SDS–PAGE and activity assay. Spontaneously inactivated LMWPTP was reactivated by incubating it with dithiothreitol (DTT), GSH, thioltransferase (TTase), and thioredoxin (TRx).To demonstrate the inactivation of LMWPTP by H₂O₂, cystine, or GSSG, DTT–reduced enzyme was incubated with oxidant for 30 min at 25°C before activity assay. In vivo studies, HLE B3 cells overexpressed with LMWPTP were exposed to H₂O₂ and the cellular LMWPTP was immuno–precipitated for activity assay. Results: Recombinant LMWPTP was purified to homogeneity with one protein band of 18 kDa, which reacted positively with anti–LMWPTP antibody. The enzyme could be inhibited completely by Na₃VO₄ (inhibitor of tyrosine phsophatase), but NaF (inhibitor of high molecular weight acid phosphatase) or EDTA (inhibitor of phosphoserine/ threonine phosphatase) was ineffective, confirming it to be LMWPTP. The enzyme was very sensitive to iodoacetamide, suggesting its cysteine residues are essential to its activity. The purified enzyme was spontaneously inactivated while in storage, however DTT could regenerate most of its activity. The enzyme could not be activated by GSH (1–10 mM) alone, but part of the activity was restored in the presence of GSH and TTase. The combination of GSH, TTase and TRx was as effective as DTT in restoring LMWPTP activity. We further demonstrated that the lens LMWPTP was extremely sensitive to H₂O₂ both in vitro and in vivo. The purified enzyme lost 25% activity when it was exposed to merely 0.1 µM H₂O₂ while no activity survived when 10 µM H₂O₂ was used. Both cystine and GSSG at 10 mM each inactivated the enzyme effectively, suggesting protein–S–S–GSH or protein–S–S–cysteine formation. H₂O₂ (0.5 mM, 30 min) could suppress 80% of the cellular LMWPTP activity, which could be regenerated by DTT. Conclusions: The activity of human lens LMWPTP could be reversibly regulated by oxidation. This is likely the mechanism how human lens epithelial cell regulates its redox signaling.

Keywords: oxidation/oxidative or free radical damage • protein modifications-post translational