Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody.
Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2(-/-) mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2(-/-) males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms.
Hoffhines
Adam J
AJ
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Damoc
Eugen
E
Bridges
Kristie G
KG
Leary
Julie A
JA
Moore
Kevin L
KL
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004822:Epididymis / Q000737:chemistry; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D051381:Rats; D013094:Spermatozoa / Q000737:chemistry; D015238:Sulfotransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011506:Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase