Different Endosomal Proteolysis Requirements for Antigen Processing of Two T-cell Epitopes of the M5 Protein from ViableStreptococcus pyogenes

Delvig, Alexei A.; Robinson, John H.

doi:10.1074/jbc.273.6.3291

Cited by 18 publications

(42 citation statements)

References 29 publications

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“…Second, the intracellular processing of CII for presentation of the glycosylated epitope was more sensitive than the nonglycosylated epitope to treatment of macrophages with inhibitors of cysteine proteinases, aspartic proteinases, and metalloproteinases, which is consistent with the interpretation that CII was differentially processed for presentation of the glycosylated and nonglycosylated epitopes. In addition, both forms of the epitope were profoundly sensitive to inhibitors of serine proteinases, which together with the results of our previous study (30), suggests a major role of this class of enzyme in antigen processing.…”

Section: Discussionsupporting

confidence: 51%

“…Adherent macrophages (10 5 /well) in 48-well flat-bottomed plates were pulsed with 20 g/ml of CII for 3 hours in the absence or presence of the enzyme inhibitors pepstatin (0.5 mM) (28), phenanthroline (0.1 mM) (29), phenylmethylsulfonyl fluoride (PMSF; 3.0 mM), 20 mM NH 4 Cl (30), (2S,3S)-trans-epoxysuccinyl-Lleucylamido-3-methylbutane ethyl ester (E-64d; 10 M) (31), N␣-p-tosyl-L-lysine chloromethylketone (TLCK; 250 M) (32), and N-p-tosyl-L-phenylalanine chloromethylketone (TPCK; 2.5 M) (29), with either cycloheximide (20 M) to block protein synthesis (33) or brefeldin A (1.0 g/ml) to disrupt Golgi transport (34). The optimal doses of inhibitors were established in separate dose-response experiments, as reported previously (30).…”

Section: Methodsmentioning

confidence: 99%

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The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

Delwig

Altmann

Isaacs

et al. 2006

Arthritis & Rheumatism

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Objective. Type II collagen (CII) is a candidate autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of CII could alter intracellular antigen processing, leading to the development of autoimmune T cell responses. To address this possibility, we studied the intracellular processing of CII for presentation of the arthritogenic glycosylated epitope CII 259-273 to CD4 T cells in macrophages from HLA-DR1-transgenic mice.Methods. HLA-DR1-transgenic mice were generated on a class II major histocompatibility complexdeficient background, and T cell hybridomas specific for the glycosylated and nonglycosylated epitope CII [259][260][261][262][263][264][265][266][267][268][269][270][271][272][273] were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype of proteinases involved.Results. We showed that the glycosylated CII 259-273epitope required more extensive processing than did the nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in the processing of CII for antigen presentation, since these compartments contained 1) enzyme activity that generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide-receptive HLA-DR1 molecules, and 4) peptide/HLA-DR1 complexes that could directly activate T cell hybridomas. Degradation of CII by dense fractions occurred optimally at pH 4.5 and was abrogated by inhibitors of serine and cysteine proteinases. Conclusion. Processing of the arthritogenic glycosylated CII259-273 epitope, which is implicated in the induction of autoimmune arthritis, is more stringently regulated than is processing of the nonglycosylated form of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel therapeutic targets for the treatment of RA.

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Section: Discussionsupporting

confidence: 51%

Section: Methodsmentioning

confidence: 99%

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

Delwig

Altmann

Isaacs

et al. 2006

Arthritis & Rheumatism

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“…The difference in the repertoire of the T cell responses that we observed as a result of this distinct Ag targeting is consistent with previous reports. Streptococcus protein Ags have been shown to require different proteases for processing and presentation of two epitopes (59), and the Ag uptake mechanism influences their presentation by determining which subcellular compartment is targeted (60). It was also reported that association of lymphocytic choriomeningitis virus minigene epitopes with another member of the LAMP protein family, lysosomal integral membrane protein II, increased the CD4 ϩ response to one epitope but decreased another, suggesting that different Ag processing pathways can indeed influence on the priming of a T cell response to a specific Ag (61).…”

Section: Discussionmentioning

confidence: 99%

Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes

Arruda

Sim²,

Chikhlikar

et al. 2006

The Journal of Immunology

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Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

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“…Treatment of APC with some inhibitors of serine proteinase activity was shown to have a limited effect on presentation of epitopes of sperm whale myoglobin (34) or hen egg lysozyme (35). However, we have demonstrated sensitivity of presentation of T-cell epitopes of the surface M protein of Streptococcus pyogenes to the chloromethyl ketones TLCK and TPCK (36), and here we have used the more effective and inclusive broad spectrum serine proteinase inhibitor DCI to implicate this enzyme family in antigen processing.…”

Section: Fig 2 the Kinetics Of Presentation Of Cd4mentioning

confidence: 99%

Differential Processing of CD4 T-cell Epitopes from the Protective Antigen of Bacillus anthracis

Musson

Walker

Flick-Smith

et al. 2003

Journal of Biological Chemistry

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We have mapped CD4؉ T-cell epitopes located in three domains of the recombinant protective antigen of Bacillus anthracis. Mouse T-cell hybridomas specific for these epitopes were generated to study the mechanisms of proteolytic processing of recombinant protective antigen for antigen presentation by bone marrow-derived macrophages. Overall, epitopes differed considerably in their processing requirements. In particular, the kinetics of presentation, ranging from 15 (fast) to 120 min (slow), suggested sequential liberation of epitopes during proteolytic processing of the intact PA molecule. Pretreatment of macrophages with ammonium chloride or inhibitors of the major enzyme families showed that T-cell responses to an epitope presented with fast kinetics were unaffected by raising endosomal pH or inhibiting cysteine or aspartic proteinases, suggesting presentation independent of lysosomal processing. In contrast, responses to epitopes presented with slower kinetics were dependent on low pH and the activity of cysteine or aspartic proteinases indicating a requirement for lysosomal processing. In addition, responses to all epitopes, whether their presentation was dependent on low pH or not, were prevented by treatment of macrophages with broad spectrum serine proteinase inhibitors. Thus, our data are consistent with a model of sequential antigen processing within the endosomal system, beginning with a pre-processing step mediated by serine or metalloproteinases prior to further processing by lysosomal enzymes. Rapidly presented epitopes seemed to require only limited proteolysis at earlier stages of endocytosis, whereas the majority of epitopes required more extensive processing by neutral proteinases followed by lysosomal enzymes.

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Different Endosomal Proteolysis Requirements for Antigen Processing of Two T-cell Epitopes of the M5 Protein from ViableStreptococcus pyogenes

Cited by 18 publications

References 29 publications

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes

Differential Processing of CD4 T-cell Epitopes from the Protective Antigen of Bacillus anthracis

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