Cloning and characterization of canine sulfotransferases

Sammanfattning: Sulfation generally leads to an increase in hydrophilicity facilitating increased excretion of conjugated molecules. In addition, sulfation has an important role in the synthesis, metabolism and regulation of steroid hormones as well as in the generation of reactive electrophilic forms of mutagenic heterocyclic amines and hydroxymethyl polyaromatic hydrocarbons. Members of the sulfotransferase (SULT) family catalyze the transfer of an electrophilic sulfonate group from the donor compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to a nucleophilic acceptor molecule. Sulfation has been studied in several species such as rat, mouse and man. However, although an important species in the study of drug metabolism, sulfation in dog has not been extensively studied. This thesis describes three different canine SULTs; cSULT1B1, cSULT1D1, and cSULT1A1 that have been cloned, expressed, and characterized regarding catalytic and kinetic properties as well as amino acid sequence and tissue distribution. The canine SULTs investigated display typical substrate specificity of the SULT1 family i.e. cSULT1B1 sulfates a-naphthol, p-nitrophenol (PNP) and thyroid hormones but not dopamine and b-estradiol (E2); cSULT1D1 sulfates a-naphthol, PNP, dopamine and serotonin but not E2; and cSULT1A1 sulfates a-naphthol, PNP, dopamine and E2. None of the cSULTs examined had detectable activity towards dehydroepiandrosterone. The tissue distribution of cSULTs was determined by immunoblot analysis. cSULT1B1 expression was readily detected in colon, kidney and small intestine of male and female dogs. A low level of expression was also present in female liver while no immunoreactivity was detected in male dog liver. cSULT1D1 expression was detected in liver, kidney, small intestine and colon and cSULT1A1 expression was detected in all the tissues tested (liver, kidney, lung, skin, small intestine, and colon). The highest expression of cSULT1B1 and cSULT1D1 was detected in colon, while that of cSULT1A1 was highest in the liver. These observations applied to both genders. In human, catecholamines such as dopamine are extensively sulfated. It appears that hSULT1A3 has evolved considerable selectivity for dopamine and other biogenic amines over phenols. One single amino acid, Glu146, was found to induce this selectivity. Similarly, cSULT1D1 was found to sulfate dopamine efficiently and in preference over PNP. To investigate the molecular basis for this selectivity in cSULT1D1, five cSULT1D1 mutants (A146E, A146D, A146Q, I86D and D247L) were engineered, expressed and characterized. The substrate preference for the A146 and I86 mutants for dopamine over PNP was unchanged, but the overall sulfation activities of these substrates were reduced. The kcat / KM value for PNP was increased over 21-fold and that of dopamine was decreased over 54-fold, hence the preference was dramatically switched from dopamine to PNP. Thus, we have identified a previously unrecognized determinant of active site selectivity in SULTs. In summary, this thesis has significantly increased the knowledge on SULTs in dog and, more generally, defined a new subfamily as well as an important determinant for substrate selectivity.