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HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules.

Denzin LK, Hammond C, Cresswell P - J. Exp. Med. (1996)

Bottom Line: HLA-DR alpha beta dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo.HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP.Incubation of peptide-free alpha beta dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, Connecticut 06510, USA.

ABSTRACT
Major histocompatibility complex (MHC) class II-positive cell lines which lack HLA-DM expression accumulate class II molecules associated with residual invariant (I) chain fragments (class II-associated invariant chain peptides [CLIP]). In vitro, HLA-DM catalyzes CLIP dissociation from class II-CLIP complexes, promoting binding of antigenic peptides. Here the physical interaction of HLA-DM with HLA-DR molecules was investigated. HLA-DM complexes with class II molecules were detectable transiently in cells, peaking at the time when the class II molecules entered the MHC class II compartment. HLA-DR alpha beta dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo. Mature, peptide-loaded DR molecules also associated at a low level. These same species, but not DR-I chain complexes, were also shown to bind to purified HLA-DM molecules in vitro. HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP. DM-DR complexes generated by incubating HLA-DM with purified DR alpha beta CLIP contained virtually no associated CLIP, suggesting that this superior interaction reflects a prolonged HLA-DM association with empty class II dimers after CLIP dissociation. Incubation of peptide-free alpha beta dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides. Stabilization of empty class II molecules may be an important property of HLA-DM in facilitating antigen processing.

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Stabilization of empty class II molecules by HLA-DM. (A and B) Loading of class II αβCLIP complexes in octyl glucoside versus loading in  octyl glucoside and DM. Purified HLA-DR3 αβCLIP complexes (25 nM) from T2.DR3 cells were incubated in 1% octyl glucoside with the DR3-specific MOMP peptide in the presence or absence of 25 nM affinity-purified HLA-DM at 37°C for the indicated times at pH 4.5. After neutralization,  samples were analyzed by SDS-PAGE (A) and the percent SDS-stable dimers formed in the presence and absence of HLA-DM at each time point were  quantitated by image analysis (B). The positions of the individual α and β chains and the αβ dimers are indicated on the right. (C and D) HLA-DM stabilizes empty class II molecules. Radiolabeled DR3 αβCLIP complexes (25 nM) were incubated at 37°C in 1% octyl glucoside in the presence and absence of HLA-DM (25 nM) for the indicated times at pH 4.5. At each time point, 1 μM MOMP peptide was added and the samples were incubated at  37°C for an additional 45 min. After neutralization, samples were analyzed by SDS-PAGE (C). To quantitate the results in panel C, the amount of SDSstable dimers at each time point is represented in panel D as the percentage of the maximal available SDS-stable dimers generated at the same time point  in the presence and absence of DM but with MOMP continuously present (B).
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Figure 5: Stabilization of empty class II molecules by HLA-DM. (A and B) Loading of class II αβCLIP complexes in octyl glucoside versus loading in octyl glucoside and DM. Purified HLA-DR3 αβCLIP complexes (25 nM) from T2.DR3 cells were incubated in 1% octyl glucoside with the DR3-specific MOMP peptide in the presence or absence of 25 nM affinity-purified HLA-DM at 37°C for the indicated times at pH 4.5. After neutralization, samples were analyzed by SDS-PAGE (A) and the percent SDS-stable dimers formed in the presence and absence of HLA-DM at each time point were quantitated by image analysis (B). The positions of the individual α and β chains and the αβ dimers are indicated on the right. (C and D) HLA-DM stabilizes empty class II molecules. Radiolabeled DR3 αβCLIP complexes (25 nM) were incubated at 37°C in 1% octyl glucoside in the presence and absence of HLA-DM (25 nM) for the indicated times at pH 4.5. At each time point, 1 μM MOMP peptide was added and the samples were incubated at 37°C for an additional 45 min. After neutralization, samples were analyzed by SDS-PAGE (C). To quantitate the results in panel C, the amount of SDSstable dimers at each time point is represented in panel D as the percentage of the maximal available SDS-stable dimers generated at the same time point in the presence and absence of DM but with MOMP continuously present (B).

Mentions: Empty class II αβ dimers have been shown to aggregate and lose their ability to bind antigenic peptides (51–53). The absence of CLIP from class II complexes coprecipitated with DM (Fig. 4 C) indicated that DM continues to interact with empty class II molecules after CLIP dissociation. Such an association might stabilize the αβ dimers and prevent them from aggregating and losing the capacity to bind antigenic peptides. To test this hypothesis, we took advantage of the ability of the detergent octyl glucoside to induce CLIP release from DR3 αβCLIP complexes (21, 27). Radiolabeled DR3 αβCLIP complexes were incubated in 1% octyl glucoside at pH 4.5 with the DR3-specific MOMP peptide in the presence and absence of affinity-purified DM. After acidification and incubation for 0 to 10 hr at 37°C, the samples were neutralized, analyzed by SDS-PAGE and quantitated by image analysis. The results showed that the conversion of class II αβCLIP complexes to αβ peptide complexes in the detergent octyl glucoside was almost as efficient in the absence of DM as in its presence. By 6 h, the percentage of SDS-stable dimers was virtually identical (∼90%; Fig. 5, A and B). This demonstrates that octyl glucoside can induce CLIP release from class II αβCLIP complexes almost as efficiently as DM under these experimental conditions. To determine whether DM could stabilize empty class II molecules, radiolabeled αβCLIP complexes were incubated without peptide in 1% octyl glucoside in the presence and absence of DM at pH 4.5 for 0–10 h. At each time point, MOMP peptide was added to 1 μM and the samples were incubated for an additional 45 min at 37°C to allow surviving, functional class II molecules to bind peptide. After neutralization, the samples were analyzed by SDS-PAGE and quantitated by image analysis. The results showed that after only 1 h of incubation in the absence of peptide more peptide-receptive class II molecules survived in the presence of DM (Fig. 5 C). With increasing incubation time, the amount of peptide-receptive class II progressively decreased both with and without DM. However, at all time points the presence of HLA-DM resulted in increased peptide loading. After 10 h of incubation no peptide-receptive class II molecules were present in the absence of DM, whereas ∼35% of class II molecules retained their peptide binding function when DM was present (Fig. 5, C and D). Control proteins at the same or 10-fold higher concentrations failed to preserve peptidereceptive class II molecules demonstrating that this function is a specific property of HLA-DM (data not shown).


HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules.

Denzin LK, Hammond C, Cresswell P - J. Exp. Med. (1996)

Stabilization of empty class II molecules by HLA-DM. (A and B) Loading of class II αβCLIP complexes in octyl glucoside versus loading in  octyl glucoside and DM. Purified HLA-DR3 αβCLIP complexes (25 nM) from T2.DR3 cells were incubated in 1% octyl glucoside with the DR3-specific MOMP peptide in the presence or absence of 25 nM affinity-purified HLA-DM at 37°C for the indicated times at pH 4.5. After neutralization,  samples were analyzed by SDS-PAGE (A) and the percent SDS-stable dimers formed in the presence and absence of HLA-DM at each time point were  quantitated by image analysis (B). The positions of the individual α and β chains and the αβ dimers are indicated on the right. (C and D) HLA-DM stabilizes empty class II molecules. Radiolabeled DR3 αβCLIP complexes (25 nM) were incubated at 37°C in 1% octyl glucoside in the presence and absence of HLA-DM (25 nM) for the indicated times at pH 4.5. At each time point, 1 μM MOMP peptide was added and the samples were incubated at  37°C for an additional 45 min. After neutralization, samples were analyzed by SDS-PAGE (C). To quantitate the results in panel C, the amount of SDSstable dimers at each time point is represented in panel D as the percentage of the maximal available SDS-stable dimers generated at the same time point  in the presence and absence of DM but with MOMP continuously present (B).
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Related In: Results  -  Collection

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Figure 5: Stabilization of empty class II molecules by HLA-DM. (A and B) Loading of class II αβCLIP complexes in octyl glucoside versus loading in octyl glucoside and DM. Purified HLA-DR3 αβCLIP complexes (25 nM) from T2.DR3 cells were incubated in 1% octyl glucoside with the DR3-specific MOMP peptide in the presence or absence of 25 nM affinity-purified HLA-DM at 37°C for the indicated times at pH 4.5. After neutralization, samples were analyzed by SDS-PAGE (A) and the percent SDS-stable dimers formed in the presence and absence of HLA-DM at each time point were quantitated by image analysis (B). The positions of the individual α and β chains and the αβ dimers are indicated on the right. (C and D) HLA-DM stabilizes empty class II molecules. Radiolabeled DR3 αβCLIP complexes (25 nM) were incubated at 37°C in 1% octyl glucoside in the presence and absence of HLA-DM (25 nM) for the indicated times at pH 4.5. At each time point, 1 μM MOMP peptide was added and the samples were incubated at 37°C for an additional 45 min. After neutralization, samples were analyzed by SDS-PAGE (C). To quantitate the results in panel C, the amount of SDSstable dimers at each time point is represented in panel D as the percentage of the maximal available SDS-stable dimers generated at the same time point in the presence and absence of DM but with MOMP continuously present (B).
Mentions: Empty class II αβ dimers have been shown to aggregate and lose their ability to bind antigenic peptides (51–53). The absence of CLIP from class II complexes coprecipitated with DM (Fig. 4 C) indicated that DM continues to interact with empty class II molecules after CLIP dissociation. Such an association might stabilize the αβ dimers and prevent them from aggregating and losing the capacity to bind antigenic peptides. To test this hypothesis, we took advantage of the ability of the detergent octyl glucoside to induce CLIP release from DR3 αβCLIP complexes (21, 27). Radiolabeled DR3 αβCLIP complexes were incubated in 1% octyl glucoside at pH 4.5 with the DR3-specific MOMP peptide in the presence and absence of affinity-purified DM. After acidification and incubation for 0 to 10 hr at 37°C, the samples were neutralized, analyzed by SDS-PAGE and quantitated by image analysis. The results showed that the conversion of class II αβCLIP complexes to αβ peptide complexes in the detergent octyl glucoside was almost as efficient in the absence of DM as in its presence. By 6 h, the percentage of SDS-stable dimers was virtually identical (∼90%; Fig. 5, A and B). This demonstrates that octyl glucoside can induce CLIP release from class II αβCLIP complexes almost as efficiently as DM under these experimental conditions. To determine whether DM could stabilize empty class II molecules, radiolabeled αβCLIP complexes were incubated without peptide in 1% octyl glucoside in the presence and absence of DM at pH 4.5 for 0–10 h. At each time point, MOMP peptide was added to 1 μM and the samples were incubated for an additional 45 min at 37°C to allow surviving, functional class II molecules to bind peptide. After neutralization, the samples were analyzed by SDS-PAGE and quantitated by image analysis. The results showed that after only 1 h of incubation in the absence of peptide more peptide-receptive class II molecules survived in the presence of DM (Fig. 5 C). With increasing incubation time, the amount of peptide-receptive class II progressively decreased both with and without DM. However, at all time points the presence of HLA-DM resulted in increased peptide loading. After 10 h of incubation no peptide-receptive class II molecules were present in the absence of DM, whereas ∼35% of class II molecules retained their peptide binding function when DM was present (Fig. 5, C and D). Control proteins at the same or 10-fold higher concentrations failed to preserve peptidereceptive class II molecules demonstrating that this function is a specific property of HLA-DM (data not shown).

Bottom Line: HLA-DR alpha beta dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo.HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP.Incubation of peptide-free alpha beta dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, Connecticut 06510, USA.

ABSTRACT
Major histocompatibility complex (MHC) class II-positive cell lines which lack HLA-DM expression accumulate class II molecules associated with residual invariant (I) chain fragments (class II-associated invariant chain peptides [CLIP]). In vitro, HLA-DM catalyzes CLIP dissociation from class II-CLIP complexes, promoting binding of antigenic peptides. Here the physical interaction of HLA-DM with HLA-DR molecules was investigated. HLA-DM complexes with class II molecules were detectable transiently in cells, peaking at the time when the class II molecules entered the MHC class II compartment. HLA-DR alpha beta dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo. Mature, peptide-loaded DR molecules also associated at a low level. These same species, but not DR-I chain complexes, were also shown to bind to purified HLA-DM molecules in vitro. HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP. DM-DR complexes generated by incubating HLA-DM with purified DR alpha beta CLIP contained virtually no associated CLIP, suggesting that this superior interaction reflects a prolonged HLA-DM association with empty class II dimers after CLIP dissociation. Incubation of peptide-free alpha beta dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides. Stabilization of empty class II molecules may be an important property of HLA-DM in facilitating antigen processing.

Show MeSH
Related in: MedlinePlus