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Biomedical subjects

A D Dunn

Publications and source records attributed to A D Dunn.

At least 19 recordsLinked to original sources
PubMed1995

Thyroids from siblings with Pendred's syndrome contain thyroglobulin messenger ribonucleic acid variants.

We studied thyroid tissue from two siblings with Pendred's syndrome (familial goiter and congenital deafness), both with the Mondini-type inner ear malformation, goiter, and hypothyroidism. Iodine trapping and peroxidase levels were grossly normal. Thyroglobulin (Tg), the only iodoprotein found, had a normal monomer size (330 kilodaltons), but low content of hormone and iodine. Tg's expected N-terminal peptides of 26 and 18 kilodaltons, usually formed in association with iodination and thyroid hormone synthesis, were absent, but appeared after iodination in vitro. Reverse transcription of ribonucleic acid from Pendred thyroid tissue and amplification by polymerase chain reaction of specific regions encoding the most important hormonogenic sites of Tg revealed a normal complementary DNA sequence corresponding to the first 100 amino acid residues in Tg's N-terminus. However, 3 of 35 clones of the 3'-region corresponding to the Tg C-terminus exhibited a deletion of nucleotides 7860-7994; this deletion was not present in any of the 150 clones from 7 other thyroids we examined. Four Pendred clones had a 2-nucleotide deletion at positions 7870-7871, a change that would result in a premature stop codon and was found in thyroids from several other subjects as well. We conclude that the messenger ribonucleic acid encoding the 3'-region of Tg can be abnormal in Pendred's syndrome. Some, but not all, of these changes also occur in other human thyroids. Further work is necessary to show if and how these alterations relate to defective hormone synthesis and goiter.

Adult↗
PubMed1991

Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L.

The normal provision of thyroid hormones to the body requires their release from the prohormone, thyroglobulin (Tg). Previous work established the importance of cathepsins B, D, and L (formerly designated cysteine proteinase I) to this process but had not defined the points of proteolytic attack for each enzyme. In the present study we labeled rabbit Tg in vivo with sodium 125I and performed limited digestions with cathepsins B, D, and L, purified from human thyroids. The resultant peptide fragments were analyzed by amino-terminal sequencing and located within the Tg molecule by comparison with the cDNA-derived sequences from human Tg. We identified three cleavage points for cathepsin B, corresponding to P'1 residues 532, 795, and 2487; four cleavage points for cathepsin L, corresponding to P'1 residues 2389, 2452, 2490, and 2657; and four cleavage points for cathepsin D, corresponding to P'1 residues 551, 1835, 2468, and 2643. None of the cleavage points was near Tgs known hormonogenic sites, but these peptide fragments contained three of the four major hormonogenic sites in rabbit Tg, suggesting some preference for their early proteolytic processing. Cathespin B alone among the three endopeptidases had some exopeptidase activity toward Tg. The cleavage specificities for each of the endopeptidases resembled those described with other protein substrates. Thus, cathepsin D preferentially cleaved bonds between hydrophobic residues, and cathespin L cleaved bonds with hydrophobic residues at P2 and P3. Although cathepsin Bs specificity was less obvious, it produced a major cleavage between 2 leucine residues. The existence of three endopeptidases cleaving at different sites shows that Tg proteolysis is a complex process, suggests synergism among their enzyme activities, and provides a physiological mechanism for selective hormone release, including its regulation by TSH.

Alkylation↗
PubMed1991

Proteolytic processing of thyroglobulin by extracts of thyroid lysosomes.

The release of T4 and T3 from the prohormone thyroglobulin (Tg) occurs in thyroid lysosomes. To examine the role of cathepsin-B, -D, and -L, the three major endopeptidases in this process, we incubated rabbit [125I]Tg, labeled in vivo, with lysosomal extracts from human thyroids. Iodopeptide formation was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after short term incubations (20-45 min), while iodoamino acid release was assessed by paper chromatography after long term incubations (8 and 24 h). Using pepstatin to inhibit cathepsin D, Z-Phe-Ala-CHN2 to inhibit both cathepsin B and L, and Z-Phe-Phe-CHN2 to selectively inhibit cathepsin L, we obtained the following results: 1) blocking of all three endopeptidases reduced both iodopeptide formation in short term experiments and iodoamino acid release in long term experiments by 80-90%; 2) iodopeptide formation was reduced by 85% with Z-Phe-Ala-CHN2, by 56% with Z-Phe-Phe-CHN2, and by 26% with pepstatin; 3) iodoamino acid release was reduced by 60-80% with Z-Phe-Ala-CHN2 and by 40-50% with either Z-Phe-Phe-CHN2 or pepstatin at 8 h, but by less than 20% at 24 h; pepstatin and Z-Phe-Phe-CHN2 together reduced iodoamino acid release by 80% and 60% at 8 and 24 h, respectively. Limited hydrolysis of Tg by lysosomal enzymes produced at least eight peptide fragments of less than 100,000 mol wt. Three of these, together representing 32% of the 125I released, resulted from cleavages in the C-terminal region of Tg corresponding to residues 2487, 2393, and 2390 of cDNA-derived human Tg. Several other peptides, together containing 38% of the 125I released, included the N-terminus of Tg. These C-terminal and N-terminal fragments contained three of Tg's four major hormonogenic sites, but none of the cleavage sites fell close to the hormone sites themselves. We conclude that 1) the formation of discrete iodopeptides precedes the release of iodothyronines and iodotyrosines from Tg; 2) the cysteine proteinases are more important than cathepsin D in this process; and 3) these endopeptidases selectively cleave Tg to favor the production of hormone-containing intermediates for subsequent processing by exopeptidases.

Chromatography, High Pressure Liquid↗
PubMed1989

The hormonogenic sites of turtle thyroglobulin and their homology with those of mammals.

Thyroglobulin (Tg) from turtles previously injected with 125I was reduced, alkylated, and digested with trypsin. We purified the resultant peptides on HPLC columns, determined their amino acid sequences and the locations of [125I]T4 and [125I]T3 residues, and compared them with established sequences from humans, cows, rabbits, rats, and guinea pigs. We found five major T4 peptides, three of which were homologous with the major hormonogenic sites A, B, and D of mammalian Tg. Site A, the highly conserved major T4 site in mammals, had substitutions in three residues near the T4 residue and had much less of Tg's newly synthesized T4 than is found in mammalian Tg (25% in turtle vs. 44% in rabbit). Site B contained correspondingly more of Tg's new T4 (42% vs. 24% in rabbit). Turtle Tg contained little [125I]T3, and we did not find site C (Ser-T3/T4-Ser, the major T3 site in guinea pig and rabbit) in turtles, but did find Val-T4, a possible homolog. Site D was quantitatively less important than in mammals. The fifth turtle hormonogenic site, containing 12% of Tg's newly formed T4, had a tyrosyl residue substituted for the phenylalanine at residue 632 in the human sequence. We conclude that Tg's major hormonogenic sites are generally conserved across a considerable evolutionary distance, but that differences in primary structure occur and may contribute to changes in priority of hormone synthesis among these sites.

Alkylation↗
PubMed1988

Thyrotropin alters the utilization of thyroglobulin's hormonogenic sites.

We injected rabbits and guinea pigs with bovine thyrotropin (TSH) daily for 3 days, while controls received saline. All animals received sodium [125I]iodide on the second day, and thyroglobulin was purified from the thyroids of each group by gel filtration. Hormonogenic tryptic peptides from each S-cyanoethylated thyroglobulin preparation were isolated by high performance liquid chromatography, and their amino acid sequences were determined, permitting their localization within the thyroglobulin polypeptide chain by comparison with cDNA-derived sequences from bovine and human thyroglobulins. Thyroglobulins from the saline-injected rabbits and guinea pigs contained the same four major hormonogenic sites, designated A-D, previously described (Dunn, J. T., Anderson, P. C., Fox, J. W., Fassler, C. A., Dunn, A. D., Hite, L. A., and Moore, R. C. (1987) J. Biol. Chem. 262, 16948-16952). In both species, sites A and C were the major loci for thyroxine and triiodothyronine, respectively. However, site D in the guinea pig had a greater ratio of [125I]thyroxine to [127I]thyroxine than did site A, whereas the reverse was true in the rabbit. TSH administration produced the following changes in thyroglobulins of both species, relative to controls: 1) an increase in the ratio of [125I]triiodothyronine to [125I] thyroxine (rabbit, 0.29 versus 0.17; guinea pig, 0.19 versus 0.08), with the increase in triiodothyronine principally at site C; 2) a marked increase in 125I/127I and in thyroxine formation at site D (14.1% of thyroglobulin's thyroxine versus 9.8% in rabbits, 24 versus 13% in guinea pigs); 3) a corresponding decrease in thyroxine formation at site A (33 versus 43% in rabbits, 30 versus 46% in guinea pigs); and 4) a sharp increase in conversion of thyroglobulin's N-terminal 125I-labeled approximately 20 kDa hormone-rich iodopeptide, which contains site A, to a 125I-labeled approximately 15-kDa (rabbit) or 125I-labeled approximately 13-kDa (guinea pig) form, reflecting probable peptide bond cleavage. Our results show that TSH alters both the structure of the thyroglobulin molecule and the priority of utilization of its hormonogenic sites. We conclude that these changes are important to TSH's enhancement of thyroid hormone synthesis.

Amino Acid Sequence↗
PubMed1988

Cysteine proteinases from human thyroids and their actions on thyroglobulin.

This report describes properties of highly purified cathepsin-B and an additional cysteine proteinase, designated cysteine proteinase I, obtained from human thyroids. Both enzymes are localized to lysosomes. The activity profile of cysteine proteinase I combined with its sensitivity to the active site inhibitor Z-Phe-Phe-CNH2 suggest that it is distinct from other cysteine proteinases described so far. Cysteine proteinase I and cathepsin-B had respective pH optima of 3.5-4.0 and 4.5-5.0 with thyroglobulin (Tg) as substrate. Based on Km/Kcat (catalytic constant) ratios, cysteine proteinase I degraded rabbit [125I]Tg to peptide intermediates 50 times more efficiently than did cathepsin-B. Under conditions of limited digestion, both enzymes cleaved Tg at three or more sites, producing iodinated fragments of 20,000-50,000 mol wt (cysteine proteinase I) or 10,000-40,000 mol wt (cathepsin-B). Tryptic digests of these fragments were isolated by HPLC, and those containing thyroid hormone were sequenced for identification of amino acids and localization of 125I. Cysteine proteinase I cleaved peptides primarily from the C-terminal region of Tg, which contained two major hormonogenic sites, while cathepsin-B produced peptides mainly from the N-terminus, containing another major hormonogenic site. We suggest that the roles of cysteine proteinase I and cathepsin-B are the rapid initial fragmentation of Tg at opposite ends of the molecule, making hormone-containing sites accessible to additional cleavage by other lysosomal endopeptidases and exopeptidases.

Caseins↗
PubMed1988

Altered immunoreactivity of thyroglobulin in thyroid disease.

We prepared 3 samples of 19S thyroglobulin (Tg), 1 from a patient with Graves' disease, another from a patient with nontoxic goiter, and the third from a pool of Tg from normal subjects, and used each Tg preparation to produce a polyvalent antiserum in rabbits. The 3 antisera were similar to each other in their reactivity with thyroid Tg samples from 25 patients with various thyroid disorders and from 10 normal subjects. However, the immunoreactivity of the 35 individual Tg samples varied considerably. Decreased reactivity was associated with proteolysis during Tg preparation, iodination in vitro with 20 or more atoms of iodine/molecule Tg, the 27S species of Tg, Tg from 3 patients with thyroid cancer, and Tg from several patients with Graves' disease. The antiserum to Graves' Tg contained some antibodies that did not bind normal Tg on an affinity column, and these antibodies reacted more with Tg from patients with Graves' disease than with Tg from normal subjects or patients with nontoxic goiters. Thus, Tg from patients with Graves' disease may contain antigenic sites that are not present or exposed in Tgs from other subjects. We conclude that thyroid Tgs from patients with Graves' disease and from those with thyroid cancer may be different in structure from the Tgs of normal subjects. This conclusion is important to an understanding of Tg structure in thyroid disease and to the use of thyroid Tg for preparation of antisera and standards for measuring serum Tg concentrations.

Animals↗
PubMed1987

The sites of thyroid hormone formation in rabbit thyroglobulin.

Rabbit thyroglobulin (Tg) was labeled in vivo with 125I and purified by gel filtration. Separation by high performance liquid chromatography (HPLC) of tryptic digests of S-cyanoethylated Tg yielded four major iodothyronine-containing peaks, designated A, B, C, and D. These were further purified on HPLC and sequenced for identification of amino acid residues and for location of the iodothyronine by 125I counting. The published primary structure for bovine Tg, derived from cDNA sequencing of the Tg gene (Mercken, L., Simons, M.J., Swillens, S., Massaer, M., and Vassart, G. (1985) Nature 316, 647-651), permitted tentative location of the rabbit hormonogenic peptides within the Tg polypeptide chain. Site A, corresponding to bovine residue 5, contained 44% of Tgs [125I]T4 (thyroxine) and 25% of its [125I]T3 (triiodothyronine); its specific activity of iodine was higher than that for other sites, indicating priority of iodination. Site B, containing 24% of Tgs [125I]T4 and 18% of its [125I]T3, corresponded to bovine residue 2555. Site C, at the third residue from the C terminus (bovine residue 2748), was the major T3 site, accounting for over 50% of Tgs [125I]T3. The amino acid sequence around this site shows less homology among different animal species than do those flanking the other hormonogenic sites. Site D accounted for 17% of Tgs [125I]T4 and corresponded to bovine Tyr-1291, in the midportion of Tgs polypeptide chain. The three major T4-forming sites had the sequence Asp-Tyr (sites B and D) or Glu-Tyr (site A), while the sequence Ser-Tyr-Ser appeared to favor T3 synthesis (site C), suggesting an important influence of primary structure on hormonogenesis. We conclude that site A is the major T4-forming site and site C the major T3-forming one, but others are available and offer the opportunity for flexibility in meeting different demands for hormone formation.

Amino Acid Sequence↗
PubMed1984

Stimulation of thyroidal thiol endopeptidases by thyrotropin.

Rabbit thyroids contain cathepsin D (CD) and several thiol endopeptidases including cathepsin B and three newly described enzymes (cathepsins 180K, 110K, and 45K). The present paper assesses the relative physiological importance of these enzymes in thyroglobulin degradation in rabbits. Thyroidal thiol endopeptidase [thiol thyroglobulin hydrolase (thiol TgH)] activity increased in the absence of changes in CD activity in animals treated with 10 U bovine TSH. Peak enzyme activity occurred 24 h after injection of hormone. After 20 U bovine TSH, thiol endopeptidase activity increased by approximately 100%, whereas CD increased by 50%. The increase in thiol enzyme activity was attributed both to cathepsin B and to the other thiol endopeptidases. The lysosomal acid hydrolases acid phosphatase and dipeptidyl peptidase II were unaffected by TSH at either dose level. Thiol TgH activity, but not CD activity, was decreased in thyroids of rabbits treated with T4 [5 micrograms/(100 g BW X day)] for 1 week. All thyroidal acid hydrolases examined were suppressed in animals receiving T4 for 3 weeks. Thiol TgH activity was localized primarily to a lysosome-enriched fraction of thyroid homogenates. Our results suggest that the thiol proteases probably are the most important endopeptidases in thyroglobulin hydrolysis in vivo and that their activities are influenced by TSH.

Animals↗
PubMed1983

The role of iodination in the formation of hormone-rich peptides from thyroglobulin.

Reduced thyroglobulins from several animal species contain hormone-rich iodopeptides of 20,000-26,000 and 15,000-18,000 daltons. The present study has investigated the role of iodination in their production. Experimental approaches have included: iodination in vitro of thyroglobulin from rabbit thyroid slices incubated with [3H]leucine and subsequent analysis of 3H distribution by gel electrophoresis; iodination in vitro of low iodine thyroglobulin from a human goiter, followed by isolation of the major iodopeptides and quantitation of their peptide content; and injection of iodine-deficient rats with Na125I and assessment of the distribution of isotope among the iodopeptides at successive time intervals. From these experiments the following general pattern has emerged: 1) at low levels of iodine (less than 5 atoms/molecule of thyroglobulin) in vitro or at short time intervals after iodine administration in vivo (less than 4 h), the principal iodinated component of reduced thyroglobulin is a approximately 230,000-dalton peptide; 2) with moderate increases in iodine (5-40 atoms/molecule) or longer time intervals after administration (greater than or equal to 4 h) there is less of the 230,000-dalton iodopeptide, and an iodothyronine-rich approximately 20,000- to 26,000-dalton iodopeptide appears; 3) at higher levels of iodine (greater than 40 atoms/molecule) the amount of approximately 230,000-dalton iodopeptide decreases further, the amount of 20,000- to 26,000-dalton iodopeptides may decrease, and an iodothyronine-rich 15,000- to 18,000-dalton peptide appears. Progressive iodination gives the same changes in distribution of peptide material among these iodopeptides as it does in iodine distribution, and the changes seen with iodination in vitro are similar to those occurring over time in vivo. We conclude that during the process of iodination discrete peptide bonds of thyroglobulin are cleaved to produce the hormone-rich iodopeptides. This is probably a normal part of thyroglobulin maturation in vivo and may be a necessary event preceding hormone formation.

Animals↗
PubMed1982

Thyroglobulin degradation by thyroidal proteases: action of purified cathepsin D.

Cathepsin D has been purified from rabbit thyroids, and its action on thyroglobulin has been examined. The enzyme was obtained in an electrophoretically homogenous form by gel filtration, followed by ion exchange chromatography and affinity chromatography with immobilized pepstatin. In some preparations, the enzyme occurred in a high molecular weight form. The ability of cathepsin D to hydrolyze [125I]thyroglobulin to fragments with a molecular weight of less than 100K was determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This activity showed a pH optimum of 3.5, was greater with reduced thyroglobulin as substrate than with the native protein, and was unaffected by potassium iodide (1-10 mM). Purified cathepsin D rapidly hydrolyzed thyroglobulin to a number of peptide intermediates. Those in the 20-45K molecular weight range had an iodothyronine content equal to or less than that of intact thyroglobulin, but the smallest peptides (apparent molecular weight, less than 2K) were iodothyronine enriched. No evidence was obtained for the release of free hormone by cathepsin D under the experimental conditions used. We conclude that cathepsin D plays a role in the initial breakdown of thyroglobulin in the thyroid and may have some selectivity for the iodothyronine portion of the molecule. The rapid hydrolysis of thyroglobulin that occurs in vivo, however, probably requires the concerted action of cathepsin D with other lysosomal endopeptidases and exopeptidases.

Animals↗
PubMed1982

Thyroglobulin degradation by thyroidal proteases: action of thiol endopeptidases in vitro.

We have obtained evidence of thiol endopeptidases in the thyroid which are active in thyroglobulin degradation in vitro. Four pepstatin-insensitive endopeptidase fractions were distinguished in extracts of rabbit thyroids by gel filtration on Bio-Gel A-0.5m. An enzyme from one fraction was obtained in highly purified form and was found to be identical to cathepsin B described in other tissues. Endopeptidases in the three remaining fractions were designated as cathepsins 180K, 110K, and 45K, respectively, on the basis of their estimated molecular size. These were partially purified by either organomercurial affinity chromatography or DEAE-cellulose chromatography. They are identified as thiol endopeptidases on the basis of their sensitivity to inhibition by both leupeptin and the thiol-blocking agent iodoacetic acid and by their activation with the reducing agent glutathione. Each is distinguished from cathepsin B on the basis of molecular size and limited ability to hydrolyze benzoylarginine-2-naphthylamide. The action of the thiol endopeptidases on [125I]thyroglobulin was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or in sodium dodecyl sulfate and urea. In each instance, the initial peptide fragments were approximately 40-45K and 30K, with iodothyronine contents similar to or less than that of intact thyroglobulin. Later products of digestion than that of intact thyroglobulin. Later products of digestion included first, 20K peptides, which showed a low iodothyronine content, and finally, peptides of approximately 10K, which showed a 1.5-fold enrichment of T4 and T3 over that of intact thyroglobulin. Each of the thiol endopeptidases had a synergistic effect when incubated with cathepsin D and [125I]thyroglobulin. Among the products of such incubations were small iodopeptides, which were iodothyronine-enriched, and free T4, itself. The results show that thiol endopeptidases are present in the thyroid gland and are collectively as important as cathepsin D in the hydrolysis of thyroglobulin in vitro. The action of these enzymes must be considered along with that of cathepsin D in understanding thyroglobulin hydrolysis in vivo.

Animals↗