cDNA cloning and expression of a new form of human aryl sulfotransferase.
To date, four human cytosolic sulfotransferases have been cloned and characterised. The aim of the present study was to identify new forms of these enzymes using molecular cloning techniques. Two full length human aryl sulfotransferase (HAST) cDNAs were cloned from a lambda gt10 liver cDNA library. The COS cell expression system was used to express the cDNAs and to determine the ability of the encoded proteins to metabolise the model substrates p-nitrophenol and dopamine. The two cDNAs were 1036 bp (HAST4) and 1060 bp (HAST4v) in length, and encoded proteins that differed by two amino acids (Thr-7 to Ile and Thr-235 to Asn). The coding domains of HAST4 and HAST4v were 97 and 94% homologous to previously reported phenol (HAST1) and monoamine (HAST3) sulfonating forms of sulfotransferase, respectively. On expression of these cDNAs in COS cells the encoded proteins were capable of sulfonating p-nitrophenol with markedly different affinities: the K(m)s for HAST4 and HAST4v being 73.7 and 7.75 microM, respectively. For the same reaction HAST1 and HAST3 have K(m)s of 0.7 and 2200 microM, respectively. Unlike HAST1 and HAST3, the expressed HAST4/4v proteins could not sulfonate dopamine. In addition to having markedly different K(m)s for p-nitrophenol as a substrate, the expressed HAST4/4 proteins also differed significantly in their affinity for the cofactor 3'-phosphoadenosine-5'-phosphosulfate. This report on the functional dissimilarity between two allelic variants of HAST4 highlights that substitution at two residues, Thr-7 and -235, markedly alters their substrate specificities and provides insight into the domains that determine these characteristics.
Zhu
X
X
Department of Physiology and Pharmacology, University of Queensland, Australia.
Veronese
M E
ME
Iocco
P
P
McManus
M E
ME
D000595:Amino Acid Sequence; D015239:Arylsulfotransferase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data
D018076:DNA, Complementary; D015239:Arylsulfotransferase