[Regulation of tyrosylprotein sulfotransferases activity by sulfotyrosine].
OBJECTIVE
To investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.


METHODS
Constructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.


RESULTS
Tyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.


CONCLUSIONS
The activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.
Gao
Jin-Ming
JM
Department of Respiratory Diseases, PUMC Hospital, CAMS and PUMC, Beijing 100730, China. gaojm@pumch.ac.cn
Feng
Qi-Ping
QP
Zuo
Jin
J
Fang
Fu-De
FD
Jiang
Lei
L
Guo
Zi-Jian
ZJ
D002460:Cell Line; D015926:Complement C3a / Q000378:metabolism; D015936:Complement C5a / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D044087:Receptor, Anaphylatoxin C5a / Q000378:metabolism; D011951:Receptors, Complement / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; C085567:complement C3a receptor; C005662:tyrosine O-sulfate; D014443:Tyrosine; D015926:Complement C3a; D015936:Complement C5a; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase