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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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(A and B) HLA-DM associates with DR in a post-Golgi  compartment. Pala cells were pulsed labeled with [35S]methionine for 15  min and chased for the indicated periods of time (h). DM-DR complexes  were immunoprecipitated at each time point from CHAPS solubilized  lysates with anti-DM (αDM). DM-associated class II molecules were  eluted with 0.5% DOC and the supernatants reprecipitated with DR β  chain-specific mAb HB10A (A) or control mAb W6/32 (B). The precipitates were either treated with Endo H or mock treated and analyzed by  SDS-PAGE (10.5%). Locations of Endo H resistant (R) and sensitive (S)  DR α and β bands are indicated on the right. (C) HLA-DM associates  with SDS-stable αβ dimers. Pala cells were pulse labeled, immunoprecipitated and re-precipitated with mAb HB10A as in A. After the addition of  Laemmli sample buffer, the precipitates were incubated at room temperature for 30 min and analyzed by SDS-PAGE (10.5%). The positions of the  individual α and β chains and the αβ dimers are indicated on the right.
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Figure 2: (A and B) HLA-DM associates with DR in a post-Golgi compartment. Pala cells were pulsed labeled with [35S]methionine for 15 min and chased for the indicated periods of time (h). DM-DR complexes were immunoprecipitated at each time point from CHAPS solubilized lysates with anti-DM (αDM). DM-associated class II molecules were eluted with 0.5% DOC and the supernatants reprecipitated with DR β chain-specific mAb HB10A (A) or control mAb W6/32 (B). The precipitates were either treated with Endo H or mock treated and analyzed by SDS-PAGE (10.5%). Locations of Endo H resistant (R) and sensitive (S) DR α and β bands are indicated on the right. (C) HLA-DM associates with SDS-stable αβ dimers. Pala cells were pulse labeled, immunoprecipitated and re-precipitated with mAb HB10A as in A. After the addition of Laemmli sample buffer, the precipitates were incubated at room temperature for 30 min and analyzed by SDS-PAGE (10.5%). The positions of the individual α and β chains and the αβ dimers are indicated on the right.

Mentions: Pulsechase analysis (Fig. 1 A) showed that DM-DR association was maximal after 1–2 h of chase, the time at which the αβI complex is delivered into the MIIC and the invariant chain is being degraded in this cell line (46; unpublished results). Therefore, the interaction must occur in a post-Golgi compartment and the DM-associated DR should be resistant to Endo H digestion. To determine this, wild-type Pala cells were pulse-labeled for 15 min with [35S]methionine and chased in the presence of an excess of methionine and cysteine. After lysis of the cells in CHAPS, the DM–class II complexes were immunoprecipitated with anti-DM. The coprecipitated DR was eluted from the DM immunoprecipitates with 0.5% DOC and reprecipitated from supernatants with a DR β chain–specific mAb, HB10A or with a negative control antibody to HLA class I molecules, W6/ 32. Duplicate HB10A and W6/32 precipitates were treated with Endo H or mock-treated overnight and analyzed by SDS-PAGE. No DM associated DR was detected early in transport since the faint Endo H–sensitive bands present immediately after the pulse in the HB10A precipitates (Fig. 2 A) were also present in the negative control precipitates (Fig. 2 B). As observed in Fig. 1, DM-associated DR was first detected after 1 h of chase and the associated DR was resistant to Endo H digestion (Fig. 2 A, note that one of the DRα chain N-linked glycans remained in the high mannose form upon maturation; 48). These data indicate that DM and DR are not associated in the ER and that DM-DR must interact after the DR molecules have traversed the medial Golgi. No HLA class I molecules were coprecipitated with DM, verifying the specificity of the DR-DM association (Fig. 2 B).


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)

(A and B) HLA-DM associates with DR in a post-Golgi  compartment. Pala cells were pulsed labeled with [35S]methionine for 15  min and chased for the indicated periods of time (h). DM-DR complexes  were immunoprecipitated at each time point from CHAPS solubilized  lysates with anti-DM (αDM). DM-associated class II molecules were  eluted with 0.5% DOC and the supernatants reprecipitated with DR β  chain-specific mAb HB10A (A) or control mAb W6/32 (B). The precipitates were either treated with Endo H or mock treated and analyzed by  SDS-PAGE (10.5%). Locations of Endo H resistant (R) and sensitive (S)  DR α and β bands are indicated on the right. (C) HLA-DM associates  with SDS-stable αβ dimers. Pala cells were pulse labeled, immunoprecipitated and re-precipitated with mAb HB10A as in A. After the addition of  Laemmli sample buffer, the precipitates were incubated at room temperature for 30 min and analyzed by SDS-PAGE (10.5%). The positions of the  individual α and β chains and the αβ dimers are indicated on the right.
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Figure 2: (A and B) HLA-DM associates with DR in a post-Golgi compartment. Pala cells were pulsed labeled with [35S]methionine for 15 min and chased for the indicated periods of time (h). DM-DR complexes were immunoprecipitated at each time point from CHAPS solubilized lysates with anti-DM (αDM). DM-associated class II molecules were eluted with 0.5% DOC and the supernatants reprecipitated with DR β chain-specific mAb HB10A (A) or control mAb W6/32 (B). The precipitates were either treated with Endo H or mock treated and analyzed by SDS-PAGE (10.5%). Locations of Endo H resistant (R) and sensitive (S) DR α and β bands are indicated on the right. (C) HLA-DM associates with SDS-stable αβ dimers. Pala cells were pulse labeled, immunoprecipitated and re-precipitated with mAb HB10A as in A. After the addition of Laemmli sample buffer, the precipitates were incubated at room temperature for 30 min and analyzed by SDS-PAGE (10.5%). The positions of the individual α and β chains and the αβ dimers are indicated on the right.
Mentions: Pulsechase analysis (Fig. 1 A) showed that DM-DR association was maximal after 1–2 h of chase, the time at which the αβI complex is delivered into the MIIC and the invariant chain is being degraded in this cell line (46; unpublished results). Therefore, the interaction must occur in a post-Golgi compartment and the DM-associated DR should be resistant to Endo H digestion. To determine this, wild-type Pala cells were pulse-labeled for 15 min with [35S]methionine and chased in the presence of an excess of methionine and cysteine. After lysis of the cells in CHAPS, the DM–class II complexes were immunoprecipitated with anti-DM. The coprecipitated DR was eluted from the DM immunoprecipitates with 0.5% DOC and reprecipitated from supernatants with a DR β chain–specific mAb, HB10A or with a negative control antibody to HLA class I molecules, W6/ 32. Duplicate HB10A and W6/32 precipitates were treated with Endo H or mock-treated overnight and analyzed by SDS-PAGE. No DM associated DR was detected early in transport since the faint Endo H–sensitive bands present immediately after the pulse in the HB10A precipitates (Fig. 2 A) were also present in the negative control precipitates (Fig. 2 B). As observed in Fig. 1, DM-associated DR was first detected after 1 h of chase and the associated DR was resistant to Endo H digestion (Fig. 2 A, note that one of the DRα chain N-linked glycans remained in the high mannose form upon maturation; 48). These data indicate that DM and DR are not associated in the ER and that DM-DR must interact after the DR molecules have traversed the medial Golgi. No HLA class I molecules were coprecipitated with DM, verifying the specificity of the DR-DM association (Fig. 2 B).

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