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Query: UMLS:C0033036 (APC)
 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is generally accepted that a limited number of T cell epitopes are generated by APC from an immunogenic protein. To ascertain the number of determinants on OVA recognized in the context of the H-2s haplotype, we generated 19 T-T hybridomas against OVA and H-2s and we synthesized 46 overlapping peptides spanning the entire protein. Eighteen T-T hybrids were stimulated by eight different peptides. The peptide recognized by one T cell hybrid was not identified. The effect of four protease inhibitors on the processing and presentation of OVA by the LS.102.9 B cell hybridoma seemed to implicate several groups of proteases in the processing of this Ag. Alkylation of cysteine residues with iodoacetic acid showed in a few cases a dramatic decrease in the capacity of OVA to stimulate T-T hybrids recognizing cysteine-free peptides. In contrast, two T-T hybrids recognizing cysteine containing peptides were not affected by the alkylation, suggesting that alkylation inhibited the processing of OVA without affecting peptide interaction with class II MHC molecules. These data demonstrate that the repertoire of peptides generated by APC from OVA is not limited to one or few immunodominant peptides, and results from the activity of several endopeptidases and/or exopeptidases. In addition, the structure of the Ag (native or denatured) was shown to affect the efficiency with which different epitopes are generated.
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PMID:Diversity in MHC class II ovalbumin T cell epitopes generated by distinct proteases. 137 66

We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.
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PMID:T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B, L, and D. 171 25

In order to examine whether the structural integrity of the hexapeptide disulfide loop (residues 17-22), present in the gamma-carboxyglutamic acid (gamma) domain of human protein C (PC), and common to all vitamin K dependent coagulation proteins, is necessary for its anticoagulant properties, we employed recombinant (r) DNA technology to generate two important variants that would address this issue. One such mutein contained aspartic acid for gamma-residue substitutions at sequence positions 19 and 20 ([gamma 19D, gamma 20D]r-PC) in the light chain of the mature protein, and the other possessed a serine for cysteine substitution at position 22 ([C22S]r-PC of the same light chain. A subpopulation of molecules of these mutant proteins, containing the maximum levels of gamma-residues in each, has been purified by fast-protein anion-exchange liquid chromatography and affinity chromatography on an anti-human PC column. A study of the kinetic characteristics of the inhibition by Ca2+ of the thrombin-catalyzed activation rates of these variants, and the corresponding stimulation by Ca2+ of the thrombin/thrombomodulin-catalyzed activation rates of the same recombinant PC molecules, demonstrated that higher concentrations of Ca2+ were required to display these effects, when compared to wild-type (wt) r-PC and human plasma PC. This suggested that the kinetically relevant Ca2+ site responsible for these effects on activation of PC, and known to be present in another domain of PC, was affected by both mutations in the gamma-domain. The recombinant PC variants were converted to their activated forms ([gamma 19D, gamma 20D]r-APC and [C22S]r-APC) and assayed for their Ca(2+)-dependent anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the hexapeptide disulfide loop present in the gamma-carboxyglutamic acid domain of human protein C in its activation properties and in the in vitro anticoagulant activity of activated protein C. 190 53

Thyroid hormonogenesis in thyroglobulin results in the conversion of an "acceptor" iodotyrosine to a hormone residue and a "donor" iodotyrosine to a dehydroalanine residue. Altogether five acceptor sites have been located as hormone residues in thyroglobulin of different animal species. To search for donor sites, we treated bovine thyroglobulin with 4-aminothiophenol to specifically modify dehydroalanine residues to S-(4-aminophenyl)cysteine (APC) residues, according to the principle of dehydroalanine determination developed by us (Kondo, T., Kondo, Y., and Ui, N. (1988) Mol. Cell. Endocr. 57, 101-106). After digesting thyroglobulin with lysyl endopeptidase, APC-containing peptides were separated from other peptides by trapping them on immobilized naphthylethylenediamine and from each other by size-exclusion and reverse-phase high performance liquid chromatography (HPLC). The HPLC patterns showed about 10 APC-containing peptides. Among them, four different peptides were purified by repeated reverse-phase HPLC. The results of partial sequencing of the four peptides by manual Edman degradation disclosed that Tyr5, Tyr926, Tyr1375, and Tyr986 or Tyr1008 are available for hormonogenesis as donor sites. These results strongly suggest that only specific tyrosine residues behave as donors.
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PMID:Location of dehydroalanine residues in the amino acid sequence of bovine thyroglobulin. Identification of "donor" tyrosine sites for hormonogenesis in thyroglobulin. 234 66

Class II MHC (Ia) molecules have been shown to be critical as restriction elements in the T helper/inducer cell recognition of antigen. Efforts to determine the role of allelic variation in MHC restricted antigen presentation have included the use of serologically selected mutants to correlate structural variations in Class II molecules with changes in the antigen presenting function of Ia bearing cells. Such studies have revealed that serologically selected mutations tend to occur in a single immunodominant region and that even a single amino acid substitution can alter T cell recognition of Ia molecules. We report here the characterization of two more serologically selected Class II A beta chain mutations. Each is due to a single base change which alters a single amino acid. One of these mutations is in the third hypervariable region (amino acid 64--glutamine to arginine) and alters the antigen presenting function. The second mutation at amino acid 48, though a relatively non-conservative change (arginine to cysteine), has no effect on APC phenotype. Such a result would be predicted based on comparisons made with the proposed three dimensional crystallographic structure of Class I molecules and models proposed for Class II molecules based on Class I structure. The amino acid change at position 48 is in a portion of the molecule that is most likely unavailable to bind antigen or interact with T cell receptor whereas the mutation at amino acid 64 is on an exposed face of the alpha helix, a region which could affect interaction with either antigen and/or the T cell receptor.
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PMID:Functional and molecular characterization of I-A kappa beta mutants is consistent with the predicted three dimensional structure of class II MHC molecules. 239 36

The helper T cell recognition of soluble globular protein antigens requires that the proteins be processed by an APC, releasing a peptide that is transported to and held on the APC surface where it is recognized by the specific T cell in conjunction with Ia. When cellular processing functions are blocked, APC lose their ability to present native antigens while retaining the capacity to activate T cells when provided with a cognate peptide fragment that contains the T cell antigenic determinant. In this report, we show that a peptide fragment of the soluble globular protein antigen tobacco hornworm moth cytochrome c, residues 92-103 containing an additional NH2-terminal cysteine residue (THMcCys92-103), is effectively presented by B cells to an I-Ek-restricted, THMc-specific T cell hybrid when covalently coupled to antibodies specific for B cell surface Ig, Ia (Ak), or class I (Kk). Maximal activation of the T cells to the THMcCys92-103-antibody conjugates is achieved with 1/100-1/1,000th of the peptide required using unconjugated THMcCys92-103 or THMcCys92-103 coupled to nonspecific antibody. The T cell response to the peptide antibody conjugates is MHC restricted, but unlike native cytochrome c-antibody conjugates, THMcCys92-103-antibody conjugates do not require processing and can be presented by paraformaldehyde-fixed B cells. The THMcCys92-103-antibody conjugate are nearly as effective when incubated with B cells, and the unbound conjugates washed away before addition of T cells as when continuously present in culture with T cells and B cells, indicating that the active peptide antibody conjugate is associated at the B cell surface. The presentation of THMcCys92-103 coupled to monovalent Fab fragments of rabbit anti-Ig antibodies is less effective than that of the peptide coupled to bivalent antibody when either live or fixed B cells are APC, indicating that the avidity for the APC surface afforded by bivalent binding may be important in the conjugate's antigenicity. The results presented here indicate that a T cell-antigenic peptide, covalently coupled to a larger antibody molecule, can be readily recognized by an Ia-restricted helper T cell in the absence of processing. Moreover, the ability of the peptide to bind to B cell surfaces greatly augments the peptide's antigenicity, even when the binding is to structures distinct from the Ia molecule required for T cell activation.
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PMID:Enhanced T cell responses to antigenic peptides targeted to B cell surface Ig, Ia, or class I molecules. 284 Apr 79

An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence. The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion. Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques. The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart. Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min. These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.
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PMID:Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli. Kinetics of processing by antigen presenting cells. 307 Mar 57

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.
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PMID:The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon. 309 42

The minor phycobiliprotein beta 16.2 was isolated from the APC-core of phycobilisomes from the cyanobacterium Mastigocladus laminosus. Its complete amino-acid sequence and some spectral characteristics are presented. beta 16.2 consists of 169 amino acids and its molecular mass is 19,390 Da. A phycocyanobilin chromophore is covalently bound to a cysteine at position 84 as in all other phycobiliproteins. The first 138 amino acids are highly homologous to the N-terminal part of beta AP (62.5%), whereas the C-terminal part has no homology. The absorption maximum is situated at 624 nm, the fluorescence emission maximum at 644 nm in phosphate buffer, pH 7.0. Both values are slightly redshifted compared with alpha AP and beta AP.
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PMID:The phycobiliprotein beta 16.2 of the allophycocyanin core from the cyanobacterium Mastigocladus laminosus. Characterization and complete amino-acid sequence. 310 45

Processing of intracellular proteins yields 8-11 residue peptides that are displayed on the APC surface as peptide/MHC class I complexes. The remarkably precise excision of antigenic peptides from their precursor polypeptides raises the question of whether specific flanking residues influence presentation efficiency. Here we addressed this question by analyzing the generation of OVA/Kb or influenza nucleoprotein/Db complexes in APC expressing precursors with varying N- or C-terminal flanking residues. We find that T cell responses were not significantly affected by varying the N-terminal flanking residue in the precursors. In contrast, presentation of peptide/MHC complexes was inhibited with the addition of a single C-terminal flanking residue. The most dramatic inhibition was observed with isoleucine, leucine, cysteine, and proline as the C-terminal flanking residues. These residue-specific variations in presentation activity could not be accounted for by differences in the stimulatory activity of corresponding synthetic peptides but were proportional to the relative amounts of naturally processed peptides recovered in the cell extracts. These findings suggest differences in the susceptibility of N- vs C-terminal flanking residues to proteolytic cleavage during Ag processing. The strong influence of specific C-terminal flanking residue(s) could be an important factor affecting the choice of peptides presented to T cells on the APC surface.
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PMID:Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. 759 93


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