De novo Sulfation of l‐Tyrosine in HepG2 Human Hepatoma Cells and Its Possible Functional Implication

Abstract

HepG2 human hepatoma cells, labeled with [35S]sulfate in the presence of 10-30 pg/ml of cycloheximide, released up to 64% of the amount of free tyrosine-0-[35S]sulfate produced and released by cells labeled in the absence of cycloheximide. A time-course study revealed that, in cells incubated in medium containing [3H]tyrosine, free [3H]tyr~sine-0-sulfate was produced within 5 min of incubation, whereas no [3H]tyrosine-sulfated proteins were detected until 20 min after the incubation had begun. Using 3'-phosphoadenosine, 5'-pho~pho[~~S]sulfate as the sulfate donor, HepG2 cell homogenate was shown to contain enzymic activity catalyzing the sulfation of L-tyrosine with the formation of tyrosine-O-["5S]sulfate. Upon subcellular fractionation, the majority of the enzyme activity was found in the cytosolic fraction. The enzyme, designated tyrosine sulfotransferase, displayed the optimum activity at pH 8.0 in the presence of 10 mM Mn2+. Under optimum conditions, the apparent K, of the enzyme for L-tyrosine, at 4.5-pM concentration of 3'-phosphoadenosine, 5'-phosphosulfate, was determined to be 1.95 mM, while that for 3'-phosphoadenosine, 5'-phosphosulfate, at 1 mM L-tyrosine concentration, was 8.3 pM. The V,,, determined under these conditions was 1.05 pmol . min-' . mg protein-'. A tyrosine-dependence study showed that, for cells labeled with [35S]sulfate, the production and release of free tyrosine-0-[35S]sulfate appeared to proceed actively and increase proportionally to the L-tyrosine concentration when it was raised above a threshold level in the culture medium. These results may imply a possible involvement of sulfation in removing excess intracellular L-tyrosine.Free tyrosine-0-sulfate (TyrS) was first discovered in human urine by Tallan et al. in 1955 [I]. Similar findings were subsequently reported for several other mammalian species [2, 31. A great number of investigations have since been made to clarify the biochemical origin of the free TyrS excreted. Studies using various cell homogenates or purified aryl sulfotransferases, however, persistently failed to reveal the enzymic activity catalyzing the sulfation of L-tyrosine [4-81. Considering the widespread occurrence of the posttranslational tyrosine sulfation among proteins and peptides of multicellular eukaryotic organisms [9], it has been proposed that the free TyrS excreted in mammalian urines represents a degradation product of tyrosine-sulfated proteins [3, 10, 111. Indeed, free T Y~ [~~S ] was shown to be generated and released when tyr~sine-[~~S]sulfated peptides or proteins were either injected into rabbit [lo] normal adult human-' [I], however, continues to raise the question concerning the necessity of the rapid turnover of tyrosine-sulfated proteins, if the latter truly is the sole source of the former. Being inspired by the recent finding that sulfation of L-tyrosine takes place in the green algae Euglena g r a d i s 1131, we decided to re-examine the possibility of the production of free TyrS via de novo sulfation of L-tyrosine in mammalian...

Purification, Characterization, and Molecular Cloning of A Novel Rat Liver Dopa/Tyrosine Sulfotransferase

“…A consensus formed (following Huttner's discovery of the widespread occurrence of the post-translational tyrosine sulfation of eukaryotic proteins (14)) is that free TyrS is generated primarily through the turnover of tyrosine sulfated proteins in vivo. We have indeed demonstrated earlier (16) that exogenous tyrosine 35 S-sulfated proteins added to the medium could be endocytosed by cultured cells and degraded intracellularly to generate free Tyr[ In our recent studies (19,20), we have obtained conclusive evidence that sulfation of L-p-tyrosine does occur in several mammalian cell lines. It is, however, unclear why mammalian cells should carry out the sulfation of an amino acid needed for protein synthesis.…”

“…Purification of the Rat Liver Dopa/Tyrosine Sulfotransferase-Preliminary experiments showed that similar to several mammalian cell lines previously studied (19,20), the Dopa/tyrosine sulfotransferase was present predominantly in the cytosolic fraction of the rat liver. The specific activity of the enzyme purified from the rat liver cytosol, with L-Dopa as the substrate, was determined to be 2,153 pmol/min/mg protein, indicating a 760-fold purification over its specific activity in the rat liver cytosol (Table I).…”

“…Section 1734 solely to indicate this fact. (19,20). We have further demonstrated that 3,4-dihydroxyphenyl-alanine (Dopa) and mtyrosine can be sulfated more efficiently by the sulfotransferase(s) present in the cytosol of HepG2 human hepatoma cells.…”

“…To convert L-p-tyrosine to L-p-TyrS, a compound destined for excretion (4), would seem to be counterproductive in terms of cellular economy. The question that should be raised then is whether L-p-tyrosine truly represents the physiological substrate of the enzyme, initially designated the "tyrosine sulfotransferase" (19). Using HepG2 human hepatoma cells as a model, we have demonstrated in a more recent study 2 that other tyrosine derivatives, e.g.…”

Sulfation of opioid drugs by human cytosolic sulfotransferases: Metabolic labeling study and enzymatic analysis

“…To date, these in vitro assay systems have revealed the sulfation of numerous compounds including tyrosine, monoamine neurotransmitters, environmental hormones as well as three opioid drugs, buprenorphine, pentazocine, and naloxone (Kurogi et al, 2012, Liu et al, 2007, Sakakibara et al, 1994, Suiko et al, 2000). HepG2 human hepatoma cell line was used as a model of hepatocyte for the metabolic labeling experiment.…”