Tyrosine 130 is an important outer ring donor for thyroxine formation in thyroglobulin.
The thyroid couples two iodotyrosine molecules to produce thyroid hormone at the acceptor site in thyroglobulin, leaving dehydroalanine or pyruvate at the donor position. Previous work has located the acceptors but not the principal iodotyrosine donors. We incorporated [14C]tyrosine into beef thyroid slices, isolated and iodinated the [14C]thyroglobulin (Tg I), separated its N-terminal approximately 22-kDa hormone-rich peptide, and digested the latter with trypsin and endoproteinase Glu-C (EC 3.4.21.19). Nonlabeled thyroglobulin (Tg II) was isolated from the same glands and processed similarly, without iodination in vitro. Tg I was used to initially recognize pyruvate in peptide fractions, and Tg II was used to then identify its location in the thyroglobulin polypeptide chain. Sequencing of a tryptic peptide by mass spectrometry and Edman degradation showed a cleavage after Val129. An endoproteinase Glu-C-generated peptide had the predicted molecular mass of a fragment containing residues 130-146 with Tyr130 replaced by pyruvate; the identification of this peptide was supported by obtaining the expected shortened fragment after tryptic digestion. 14C-labeled pyruvate was identified in the same fraction as this peptide. We conclude that Tyr130 is an important donor of the outer iodothyronine ring. Its likely acceptor is Tyr5, the most important hormonogenic site of thyroglobulin, because Tyr5 and Tyr130 are proximate, because they are the most prominent early iodination sites in this part of thyroglobulin, and because the N-terminal region was previously found capable of forming T4 by itself.
Dunn
A D
AD
Division of Endocrinology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. add7k@virginia.edu
Corsi
C M
CM
Myers
H E
HE
Dunn
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D013954:Thyroglobulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013974:Thyroxine / Q000096:biosynthesis*; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D013954:Thyroglobulin; D013974:Thyroxine