Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases.
Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.
Niehrs
C
C
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Huttner
W B
WB
Carvallo
D
D
Degryse
E
E
D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D002417:Cattle; D002766:Cholecystokinin / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism*; D007700:Kinetics; D007865:Leeches / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013431:Sulfates; C005662:tyrosine O-sulfate; D014443:Tyrosine; D010724:Phosphoadenosine Phosphosulfate; D002766:Cholecystokinin; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D013917:Thrombin