The goals of this project are: 1) A detailed understanding of the structure and function of the thyrogobulin molecule. 2) The definition of the process of thyroid hormone biosynthesis and the factors which regulate it. 3) An examination of the interaction of thyroglobulin with thyroid peroxidase. The data obtained in these studies will contribute to an understanding of the process of thyroid hormonogenesis and in turn, to an appreciation of those factors which are responsible for various abnormal thyroid states. Such understanding should also facilitate new approaches to the diagnosis and clinical management of thyroid disease. This research continues an effort to identify and characterize those amino acid sequences within thyroglobulin which are sites of post-translational modification. These include sites which are iodinated by thyroid peroxidase and ultimately coupled to produce thyroid hormone as well as sites of glycosylation, phosphorylation, and if a recent observation holds, sulfation. Two key approaches in this proposal are: 1) The isolation and characterization of a series of naturally occurring thyroxine containing polypeptides derived from thyroglobulin upon reduction and alkylation of disulfide bonds and 2) The separation of tryptic peptides derived from thyroglobulin or its subfragments using HPLC. The latter has greatly facilitated the isolation of specific peptides in sufficient quantities to allow amino acid sequence determination. Amino acid sequence information is being correlaed with nucleotide sequence data being developed in several laboratories. The origin of the naturally occurring polypeptides is under investigation. Peptides which contain dehydroalanine, the proposed by-product of hormone formation, will be identified and isolated using specific labeling and heir amino acid sequence determined. Tryptic glycopeptides will be isolated and sequenced in order to define the sites of glycosylation with the primary structure predicted by thyroglobulin cDNA sequence. In addition, thyroglobulin will be prepared in vitro thyroid slice experiments labeled with radioactive isotopes of phosphate and sulfate respectively. This radioactivity will be used to identify peptides containing sites of either sulfation or phosphorylation in HPLC maps. These peptides will be isolated and their sequence determined in order to allow the placement of additional sites of post translation modification within the predicted sequence determined from cDNA. HPLC peptide mapping procedures and will be applied to small tissue samples obtained from abnormal and normal human thyroid tissues obtained incident to thyroid surgery or autopsy. Peptide maps, as well as iodine distribution within the peptides, will be studied as a function of disease as well as age and sex. The structure and function of thyroid peroxidase are being studied along with new methodology for its purification.
