Limits...
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.

Show MeSH

Related in: MedlinePlus

The 34-45, 46-61, and 116-129 determinants in HEL all require endocytic processing for presentation, but only 46-61 requires  newly synthesized proteins. B lymphoblasts were incubated with the indicated concentrations of HEL in standard medium for 6 h or with 50 μM  HEL for 6 h in the presence of the indicated concentrations of drug. The  B cells were then fixed and used as APCs with T hybridomas specific for  each determinant. IL-2 production at 24 h is presented as OD obtained  using an IL-2–specific capture ELISA. The data represent one of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196034&req=5

Figure 1: The 34-45, 46-61, and 116-129 determinants in HEL all require endocytic processing for presentation, but only 46-61 requires newly synthesized proteins. B lymphoblasts were incubated with the indicated concentrations of HEL in standard medium for 6 h or with 50 μM HEL for 6 h in the presence of the indicated concentrations of drug. The B cells were then fixed and used as APCs with T hybridomas specific for each determinant. IL-2 production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of three independent experiments.

Mentions: Many previous studies have shown varying sensitivities of class II antigen presentation to metabolic inhibitors, or distinct requirements for particular cell types, for new protein synthesis, or for Ii, suggesting the existence of multiple distinct pathways for antigen processing and presentation by class II molecules. Most of these, however, have involved comparisons between different proteins or distinct MHC class II alleles. To more systematically explore this issue, we have turned to a well-studied model antigen. HEL contains three distinct determinants (34-45, 46-61, and 116-129) that bind to the same class II molecule, AαkAβk, with a different dependence on the coexpression of Ii (19) or lysosome-dependent disulfide bond reduction and fragmentation of the HEL protein (44, 45). We first verified that all three determinants require pH -sensitive intracellular processing (Fig. 1). The lysosomotropic amine chloroquine inhibits the presentation of all three determinants by normal B cell blasts (Fig. 1 B). Furthermore, if these B cell APCs are chemically fixed before antigen exposure, they present synthetic peptides and denatured HEL, but not intact, nondenatured HEL (data not shown). These results indicate that T cell recognition of all three determinants requires active intracellular processing of the native protein. Despite this, when BFA is used to block export of newly synthesized proteins from the endoplasmic reticulum, only the presentation of HEL 46-61 is strongly inhibited (Fig. 1 C). This suggests that presentation of HEL 34-45 and 116-129 may involve a pool of mature class II molecules.


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)

The 34-45, 46-61, and 116-129 determinants in HEL all require endocytic processing for presentation, but only 46-61 requires  newly synthesized proteins. B lymphoblasts were incubated with the indicated concentrations of HEL in standard medium for 6 h or with 50 μM  HEL for 6 h in the presence of the indicated concentrations of drug. The  B cells were then fixed and used as APCs with T hybridomas specific for  each determinant. IL-2 production at 24 h is presented as OD obtained  using an IL-2–specific capture ELISA. The data represent one of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The 34-45, 46-61, and 116-129 determinants in HEL all require endocytic processing for presentation, but only 46-61 requires newly synthesized proteins. B lymphoblasts were incubated with the indicated concentrations of HEL in standard medium for 6 h or with 50 μM HEL for 6 h in the presence of the indicated concentrations of drug. The B cells were then fixed and used as APCs with T hybridomas specific for each determinant. IL-2 production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of three independent experiments.
Mentions: Many previous studies have shown varying sensitivities of class II antigen presentation to metabolic inhibitors, or distinct requirements for particular cell types, for new protein synthesis, or for Ii, suggesting the existence of multiple distinct pathways for antigen processing and presentation by class II molecules. Most of these, however, have involved comparisons between different proteins or distinct MHC class II alleles. To more systematically explore this issue, we have turned to a well-studied model antigen. HEL contains three distinct determinants (34-45, 46-61, and 116-129) that bind to the same class II molecule, AαkAβk, with a different dependence on the coexpression of Ii (19) or lysosome-dependent disulfide bond reduction and fragmentation of the HEL protein (44, 45). We first verified that all three determinants require pH -sensitive intracellular processing (Fig. 1). The lysosomotropic amine chloroquine inhibits the presentation of all three determinants by normal B cell blasts (Fig. 1 B). Furthermore, if these B cell APCs are chemically fixed before antigen exposure, they present synthetic peptides and denatured HEL, but not intact, nondenatured HEL (data not shown). These results indicate that T cell recognition of all three determinants requires active intracellular processing of the native protein. Despite this, when BFA is used to block export of newly synthesized proteins from the endoplasmic reticulum, only the presentation of HEL 46-61 is strongly inhibited (Fig. 1 C). This suggests that presentation of HEL 34-45 and 116-129 may involve a pool of mature class II molecules.

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