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Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein.

Zhong G, Romagnoli P, Germain RN - J. Exp. Med. (1997)

Bottom Line: Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary.Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway.This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

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Differential presentation of the three HEL determinants by  IFN-γ–activated macrophages and the effects of inhibitors of protein degradation on this presentation. Peritoneal macrophages were cultured in IFN-γ  for 48 h, and then exposed to the indicated concentrations of HEL for 6 h  or 50 μM HEL for 6 h with or without the indicated drugs. Cells were  then washed, fixed, and used as APCs for the appropriate T hybridomas. IL-2  production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of the three independent experiments.
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Figure 6: Differential presentation of the three HEL determinants by IFN-γ–activated macrophages and the effects of inhibitors of protein degradation on this presentation. Peritoneal macrophages were cultured in IFN-γ for 48 h, and then exposed to the indicated concentrations of HEL for 6 h or 50 μM HEL for 6 h with or without the indicated drugs. Cells were then washed, fixed, and used as APCs for the appropriate T hybridomas. IL-2 production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of the three independent experiments.

Mentions: The 34-45, 46-61, and 116-129 determinants of HEL lie in very different locations in the folded structure of the intact HEL protein, with 116-129 being most superficial, 34-45 being partially buried, and 46-61 lying at the core of the protein in a structure maintained by disulfide bonds. Presentation of the 46-61 determinant of HEL requires the reducing environment found in lysosomes (44, 45) and this correlates with where newly synthesized class II–Ii complexes accumulate before Ii digestion and CLIP removal (10, 47–51). On the other hand, the inability of Ii to rescue presentation of 116-129 in cells expressing tail-mutated class II suggests that this determinant may not be available in such highly acidic, dense, endocytic organelles. In this regard, Frosch et al. have reported that IFN-γ activated MΦ fail to present a particular insulin determinant (52), although B cells from the same mouse are fully competent to do so. This difference appears to be related to the more rapid and complete digestion of insulin by the MΦ, which prevents effective capture by class II. Given the different locations of the three HEL determinants in the native protein, one might expect that 116-129 and perhaps 34-45, but not 46-61, to be overdigested in activated macrophages, as compared to the B cell blasts used in the studies shown in Fig. 1. As shown in Fig. 6, this is the case. IFN-γ–activated macrophages are quite effective in presenting the 46-61 determinant, but inefficient in presenting either the 34-45 or 116-129 determinants. If chloroquine or leupeptin is added to HEL-exposed macrophages before fixation, there is a paradoxical dose-dependent gain in presentation of the 34-45 and 116-129 determinants, but a decline in the presentation of 46-61.


Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein.

Zhong G, Romagnoli P, Germain RN - J. Exp. Med. (1997)

Differential presentation of the three HEL determinants by  IFN-γ–activated macrophages and the effects of inhibitors of protein degradation on this presentation. Peritoneal macrophages were cultured in IFN-γ  for 48 h, and then exposed to the indicated concentrations of HEL for 6 h  or 50 μM HEL for 6 h with or without the indicated drugs. Cells were  then washed, fixed, and used as APCs for the appropriate T hybridomas. IL-2  production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of the three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196034&req=5

Figure 6: Differential presentation of the three HEL determinants by IFN-γ–activated macrophages and the effects of inhibitors of protein degradation on this presentation. Peritoneal macrophages were cultured in IFN-γ for 48 h, and then exposed to the indicated concentrations of HEL for 6 h or 50 μM HEL for 6 h with or without the indicated drugs. Cells were then washed, fixed, and used as APCs for the appropriate T hybridomas. IL-2 production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of the three independent experiments.
Mentions: The 34-45, 46-61, and 116-129 determinants of HEL lie in very different locations in the folded structure of the intact HEL protein, with 116-129 being most superficial, 34-45 being partially buried, and 46-61 lying at the core of the protein in a structure maintained by disulfide bonds. Presentation of the 46-61 determinant of HEL requires the reducing environment found in lysosomes (44, 45) and this correlates with where newly synthesized class II–Ii complexes accumulate before Ii digestion and CLIP removal (10, 47–51). On the other hand, the inability of Ii to rescue presentation of 116-129 in cells expressing tail-mutated class II suggests that this determinant may not be available in such highly acidic, dense, endocytic organelles. In this regard, Frosch et al. have reported that IFN-γ activated MΦ fail to present a particular insulin determinant (52), although B cells from the same mouse are fully competent to do so. This difference appears to be related to the more rapid and complete digestion of insulin by the MΦ, which prevents effective capture by class II. Given the different locations of the three HEL determinants in the native protein, one might expect that 116-129 and perhaps 34-45, but not 46-61, to be overdigested in activated macrophages, as compared to the B cell blasts used in the studies shown in Fig. 1. As shown in Fig. 6, this is the case. IFN-γ–activated macrophages are quite effective in presenting the 46-61 determinant, but inefficient in presenting either the 34-45 or 116-129 determinants. If chloroquine or leupeptin is added to HEL-exposed macrophages before fixation, there is a paradoxical dose-dependent gain in presentation of the 34-45 and 116-129 determinants, but a decline in the presentation of 46-61.

Bottom Line: Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary.Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway.This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

Show MeSH
Related in: MedlinePlus