Generation and release of nitrotyrosine O-sulfate by HepG2 human hepatoma cells upon SIN-1 stimulation: identification of SULT1A3 as the enzyme responsible

Abstract

In addition to serving as a biomarker of oxidative/nitrative stress, elevated levels of nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. Whether the body is equipped with mechanisms for protecting against the potentially harmful nitrotyrosine remains unknown. The present study was designed to investigate the possibility that sulfation serves as a pathway for the metabolism/regulation of nitrotyrosine. Using metabolic labelling, nitrotyrosine O-[35S]sulfate was found to be produced and released into the medium of HepG2 human hepatoma cells labelled with [35S]sulfate in the presence of nitrotyrosine. To identify the enzyme(s) responsible for nitrotyrosine sulfation, a systematic study of all eleven known human cytosolic SULTs (sulfotransferases) was performed. Of the 11 enzymes tested, only SULT1A3 displayed sulfating activity toward nitrotyrosine. The pH-dependence and kinetic constants of SULT1A3 with nitrotyrosine or dopamine as substrate were determined. To examine whether the sulfation of nitrotyrosine occurs in the context of cellular physiology, HepG2 cells labelled with [35S]sulfate were treated with SIN-1 (morpholinosydnonimine), a peroxynitrite generator. Increments of nitrotyrosine O-[35S]sulfate were detected in the medium of HepG2 cells treated with higher concentrations of SIN-1. To gain insight into the physiological relevance of nitrotyrosine sulfation, a time-course study was performed using [3H]tyrosine-labelled HepG2 cells treated with SIN-1. The findings confirm that the bulk of free [3H]nitrotyrosine inside the cells was present in the unconjugated form. The proportion of sulfated [3H]nitrotyrosine increased dramatically in the medium over time, implying that sulfation may play a significant role in the metabolism of free nitrotyrosine.

Figure 1. Identification of free nitrotyrosine O-[³⁵S]sulfate generated and released by HepG2 human hepatoma cells metabolically labelled with [³⁵S]sulfate in the presence of nitrotyrosine

Autoradiograph taken from the TLC plate used for the TLE analysis. Confluent HepG2 cells were incubated in the labelling media containing 0, 5, 10, 25 or 50 μM nitrotyrosine for 18 h as indicated. The broken-line circles indicate the position of nitrotyrosine O-[³⁵S]sulfate (NitroTyrS).

Figure 2. pH-dependence of the sulfating activity of human SULT1A3 with (A) nitrotyrosine and (B) dopamine as substrates

The enzymatic assays were carried out under standard assay conditions as described in the Experimental section, using different buffer systems. Results are means±S.D. for three experiments.

Figure 3. Lineweaver–Burk double-reciprocal plots of SULT1A3 with L-p-tyrosine as substrate in the absence or presence of different concentrations of nitrotyrosine

Concentrations of nitrotyrosine tested were 0 (□), 125 (■), 250 (▲) and 500 (●) μM. Concentrations of L-p-tyrosine were in mM and velocities were expressed as nmol of sulfated product formed/min per mg of protein. Results are means for three determinations. The inset shows an enlargement of the region where the lines converge in the plot.

Figure 4. Generation and release of free nitrotyrosine O-[³⁵S]sulfate by HepG2 human hepatoma cells labelled with [³⁵S]sulfate in the presence of SIN-1

Autoradiograph taken from the TLC plate used for the TLE analysis. Confluent HepG2 cells were incubated for 18 h in the labelling media containing 0, 0.5, 1 and 2.5 mM SIN-1 (lanes 2–5 respectively). Lane 1 shows the nitrotyrosine O-[³⁵S]sulfate generated in the enzymatic assay. The broken-line circles indicate the position of nitrotyrosine O-[³⁵S]sulfate (NitroTyrS).

Figure 5. Identification of free nitrotyrosine O-[³⁵S]sulfate and tyrosine O-[³⁵S]sulfate generated and released by HepG2 human hepatoma cells

Autoradiograph taken from the TLC plate used for the two-dimensional thin-layer analysis of the labelling medium sample. Confluent HepG2 cells were incubated for 18 h in the labelling media containing 2.5 mM SIN-1. The broken-line circles correspond to the positions of synthetic nitrotyrosine O-sulfate (upper) and tyrosine O-sulfate (lower) as revealed by ninhydrin staining.