Selectivity in tyrosyl iodination sites in human thyroglobulin.
Previous studies indicate that when low iodine thyroglobulin (Tg) is iodinated enzymatically with thyroid peroxidase (TPO), the tyrosyl residues that are used for the formation of thyroid hormone (hormonogenic sites) are selected for early iodination. The aim of the present study was to assess the relative importance of the substrate (Tg) and the enzyme (TPO) in the selection of the early tyrosyl sites that undergo iodination. For this purpose, low iodine human Tg (2.0 atoms I per 660,000 dimer) was iodinated chemically with (125)I-(3) and enzymatically with TPO + 125I- to a matched low level of iodination (approximately 8 added I atoms per molecule). After reduction and alkylation, the two Tg preparations were digested with trypsin, and the tryptic digests were separated by reverse-phase HPLC into 10 125I-containing pools. Each pool was further fractionated by HPLC to provide purified 125I-peptides suitable for sequence analysis. From the sequence information and the known amino acid sequence of Tg, it was possible to define the location of the iodinated tyrosyl residues. Surprisingly, almost identical results were obtained with chemically and enzymatically iodinated Tg. Not only were the 125I-peptide maps very similar, but all of the recovered 125I in the purified peptides from both samples was located in only three different tyrosyl sites, 5, 2553, and 2520. Tyr 5 and Tyr 2553 are well-established sites of thyroxine formation, while Tyr 2520 has previously been proposed by us to be a donor site. Our observation that the same hormonogenic tyrosyl sites are iodinated by chemical as well as enzymatic iodination indicates that preferential iodination of hormonogenic sites is dependent primarily on the native structure of Tg. TPO plays a minor role, if any, in the selection of early tyrosyl iodination sites in Tg. Consistent with this conclusion was our finding that chemical iodination, as well as enzymatic iodination, led to formation of uniformly iodinated Tg, as determined by isopycnic centrifugation in rubidium chloride. However, we observed a slightly higher diiodotyrosine (DIT) content and a correspondingly lower monoiodotyrosine content in enzymatically iodinated Tg, compared to matched chemically iodinated Tg. This was not observed with two other proteins, bovine serum albumin and trypsinogen, or with free tyrosine, as substrates for iodination. The same preferential formation of DIT in Tg was, however, observed when lactoperoxidase was substituted for TPO. Preferential formation of DIT, therefore, appears to involve interaction between Tg and the peroxidase.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160, USA.
S
S
Xiao
M L
ML
Dorris
A B
AB
Rawitch
A
A
Taurog
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005340:Fibrinogen / Q000737:chemistry; D005612:Freeze Drying; D006042:Goiter / Q000378:metabolism; D006801:Humans; D007453:Iodide Peroxidase / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013552:Swine; D013954:Thyroglobulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D005340:Fibrinogen; D013954:Thyroglobulin; D007453:Iodide Peroxidase; D014357:Trypsin