Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.
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PMID:T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT). 128 74

Macrophage uptake and presentation of thyroglobulin (TG) were investigated in rats with experimental autoimmune thyroiditis induced by immunization with TG and adjuvant. Peritoneal macrophages were shown to take up radiolabelled TG and this did not depend on the presence of thyroiditis in the animal or on the strain of rat used. Cell-associated antigen was largely unaffected by trypsin. Macrophages primed in vitro with TG produced a rise in the circulating TG autoantibody levels of convalescent animals when given intravenously (unlike free antigen), but did not induce disease in virgin animals. These findings show that macrophages can bind and present TG and suggest that this process may perpetuate otherwise transient autoimmune reactions.
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PMID:Thyroglobulin uptake and presentation by macrophages in experimental autoimmune thyroiditis. 661 16

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37 degrees C using non-reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides, the largest of which with an apparent molecular weight (MWap) of 40 kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18-24 h of incubation. Tryptic peptides of Tg, released at 1 h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti-Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWap of 10-25 kD and above. Four other MoAbs reacted with peptides of MWap of 25-43 kD and above, and the remaining four reacted with peptides of MWap > 43 kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation-dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.
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PMID:Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies. 752 42

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.
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PMID:Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases. 1034 9

Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the humoral autoimmune response in human autoimmune thyroiditis (Hashimoto's thyroiditis). The majority of TPO autoantibodies in individual patients' sera interact with a restricted immunodominant region on TPO. Although this region can be mapped, previous studies have failed to localize its position on the TPO molecule. We, therefore, used a footprinting approach that can localize a highly conformational, discontinuous epitope on a very large molecule. Extensive biotinylation ( approximately 15 biotins/molecule protein) of lysine residues on the surface of purified, native TPO resulted in loss of multiple tryptic cleavage sites, as determined by analysis of tryptic polypeptide fragments on reverse-phase HPLC. TPO was then complexed with a monoclonal human autoantibody Fab (TR1.9) before biotinylation. After dissociation from TR1.9, TPO was recovered by gel filtration. A trypsin site, previously observed to be lost after TPO biotinylation, was restored when biotinylation was performed on the TPO-TR1.9 complex. The epitope-protected lysine (K) was present in a 30-aa TPO fragment that, by N-terminal sequencing, was found to be K713. Altered recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule involving this region supported the epitope protection data. In conclusion, we provide the first identification of an amino acid residue (K713) comprising part of an epitope within the TPO immunodominant region. This focal residue localizes the facet on the large, highly complex TPO molecule that contains the immunodominant region and provides the basis for rational guided mutagenesis studies to more fully characterize this region.
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PMID:Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope. 1114 17

Oral tolerance is the mechanism by which the immune system remains unresponsive to orally administered soluble antigens. Mice immunized with human TG (hTG), resulting in the induction of experimental autoimmune thyroiditis (EAT), provide an ideal in vivo system in which to examine oral tolerance to hTG. In the present study, we characterize epitopes of hTG that are capable of inducing oral tolerance. hTG is a large homodimeric protein, 660 Kd. The limited proteolysis of hTG using trypsin (TR) generates several smaller fragments of hTG ranging in size from 29 Kd to 145 Kd. Using hTG fragments h1TR (residues 1-521), h4bisTR (residues 2513-2713), h6TR (residues 522-1626), and h7TR (residues 1627-2512), prepared from both iodine rich and iodine poor hTG, we investigated the ability of these fragments to induce oral tolerance. The oral administration of iodine rich h6TR or h7TR suppresses hTG specific immune responses in a manner similar to whole hTG. In contrast, the oral administration of iodine rich h1TR or h4bisTR exacerbates hTG specific immune responses. Unlike iodine rich h1TR or h4bisTR, the oral administration of iodine poor h1TR or h4bisTR fails to augment hTG specific immune responses. In fact, h4bisTR suppresses hTG specific immune responses. These results indicate that hTG contains multiple epitopes that differentially affect oral tolerization. Tolerogenic epitopes reside within fragments h6TR and h7TR. The removal of iodine, and presumably hormone, from h4bisTR converts an immunogenic epitope to a tolerogenic epitope.
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PMID:Multiple fragments of human TG are capable of inducing oral tolerance to whole human TG. 1284 35