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Intermediates in the assembly and degradation of class I major histocompatibility complex (MHC) molecules probed with free heavy chain-specific monoclonal antibodies.

Machold RP, Ploegh HL - J. Exp. Med. (1996)

Bottom Line: Shortly after completion of the polypeptide chain, reactivity with KU1, KU2, and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process.Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive K(b) heavy chains.Thus, free heavy chains that arise at the cell surface are destroyed after internalization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before assembly with peptide and beta 2-microglobulin, and later, upon disintegration of the heterotrimeric complex. To characterize the structures of folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies have been produced that recognize epitopes along the H-2K(b) heavy chain which are obscured upon proper folding and subsequent assembly with beta 2-microglobulin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193-198). The K(b) heavy chain is inserted into the lumen of the endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2, and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process. Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive K(b) heavy chains. At the cell surface, a pool of free K(b) heavy chains appears after 60-120 min of chase, whose subsequent degradation, but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free heavy chains that arise at the cell surface are destroyed after internalization.

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The monoclonal antibodies KU1, KU2, and KU4 are specific for free heavy chains. (A) mRNAs for H-2Kb and mouse β2m were translated  in vitro for 60 min, following which class I molecules were immunoprecipitated with the anti-H-2 antiserum, or the monoclonal antibodies KU1, KU2,  and KU4, before analysis by SDS–PAGE. (B) RMA cells were pulsed for 1 min and chased for the times indicated. Aliquots of cells were lysed directly in  digitonin lysis mix containing either Y3 (class I complexes), Raf  HC (free heavy chains), KU2, or α-calnexin. Before SDS–PAGE analysis, the immunoprecipitated class I material was reimmunoprecipitated with p8.
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Figure 3: The monoclonal antibodies KU1, KU2, and KU4 are specific for free heavy chains. (A) mRNAs for H-2Kb and mouse β2m were translated in vitro for 60 min, following which class I molecules were immunoprecipitated with the anti-H-2 antiserum, or the monoclonal antibodies KU1, KU2, and KU4, before analysis by SDS–PAGE. (B) RMA cells were pulsed for 1 min and chased for the times indicated. Aliquots of cells were lysed directly in digitonin lysis mix containing either Y3 (class I complexes), Raf  HC (free heavy chains), KU2, or α-calnexin. Before SDS–PAGE analysis, the immunoprecipitated class I material was reimmunoprecipitated with p8.

Mentions: RMA cells or Con A-stimulated splenocytes were starved in RPMI-1640 medium lacking methionine and cysteine for 45 min before being pulsed with 500 μCi/ml [35S]methionine/cysteine (80:20) for the times indicated. When included, DTT (5 mM) was added 5 min before labeling, and the inhibitors N-7-oxadecyl-dNM (7-0-dec; 2 mM) and concanamycin B (Con B; 20 nM) were added during the starvation period. Labeling was terminated by adding 1 mM cold methionine/cysteine to the cell suspension. For the short (1–2 min) pulse–chase analyses (see Figs. 3 and 4), aliquots of cells (2–3 × 106) were removed at each timepoint and directly lysed in ice cold digitonin lysis buffer (0.5% digitonin [Sigma], 25 mM Hepes, pH 7.2, 10 mM CaCl2, 1 mM PMSF, 10 mM iodoacetamide) containing mAb or antiserum. After centrifugation of the cell lysates to remove nuclei and cellular debris, immune complexes were isolated after a 2-h incubation (with agitation) at 4°C by incubation with 50 μl 10% fixed Staphylococcus aureus for an additional 45 min. The S. aureus pellets were washed once in ice cold digitonin lysis buffer and then boiled for 10 min in denaturation/reduction buffer (2% SDS, 5 mM dithiothreitol, 50 mM Tris/HCl, pH 7.8, 1 mM EDTA). After one preclearing step with normal rabbit serum, the eluted Kb class I heavy chains were then reimmunoprecipitated in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 1 mM PMSF, 10 mM iodoacetamide) with the anti-p8 antiserum before gel analysis.


Intermediates in the assembly and degradation of class I major histocompatibility complex (MHC) molecules probed with free heavy chain-specific monoclonal antibodies.

Machold RP, Ploegh HL - J. Exp. Med. (1996)

The monoclonal antibodies KU1, KU2, and KU4 are specific for free heavy chains. (A) mRNAs for H-2Kb and mouse β2m were translated  in vitro for 60 min, following which class I molecules were immunoprecipitated with the anti-H-2 antiserum, or the monoclonal antibodies KU1, KU2,  and KU4, before analysis by SDS–PAGE. (B) RMA cells were pulsed for 1 min and chased for the times indicated. Aliquots of cells were lysed directly in  digitonin lysis mix containing either Y3 (class I complexes), Raf  HC (free heavy chains), KU2, or α-calnexin. Before SDS–PAGE analysis, the immunoprecipitated class I material was reimmunoprecipitated with p8.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211561&req=5

Figure 3: The monoclonal antibodies KU1, KU2, and KU4 are specific for free heavy chains. (A) mRNAs for H-2Kb and mouse β2m were translated in vitro for 60 min, following which class I molecules were immunoprecipitated with the anti-H-2 antiserum, or the monoclonal antibodies KU1, KU2, and KU4, before analysis by SDS–PAGE. (B) RMA cells were pulsed for 1 min and chased for the times indicated. Aliquots of cells were lysed directly in digitonin lysis mix containing either Y3 (class I complexes), Raf  HC (free heavy chains), KU2, or α-calnexin. Before SDS–PAGE analysis, the immunoprecipitated class I material was reimmunoprecipitated with p8.
Mentions: RMA cells or Con A-stimulated splenocytes were starved in RPMI-1640 medium lacking methionine and cysteine for 45 min before being pulsed with 500 μCi/ml [35S]methionine/cysteine (80:20) for the times indicated. When included, DTT (5 mM) was added 5 min before labeling, and the inhibitors N-7-oxadecyl-dNM (7-0-dec; 2 mM) and concanamycin B (Con B; 20 nM) were added during the starvation period. Labeling was terminated by adding 1 mM cold methionine/cysteine to the cell suspension. For the short (1–2 min) pulse–chase analyses (see Figs. 3 and 4), aliquots of cells (2–3 × 106) were removed at each timepoint and directly lysed in ice cold digitonin lysis buffer (0.5% digitonin [Sigma], 25 mM Hepes, pH 7.2, 10 mM CaCl2, 1 mM PMSF, 10 mM iodoacetamide) containing mAb or antiserum. After centrifugation of the cell lysates to remove nuclei and cellular debris, immune complexes were isolated after a 2-h incubation (with agitation) at 4°C by incubation with 50 μl 10% fixed Staphylococcus aureus for an additional 45 min. The S. aureus pellets were washed once in ice cold digitonin lysis buffer and then boiled for 10 min in denaturation/reduction buffer (2% SDS, 5 mM dithiothreitol, 50 mM Tris/HCl, pH 7.8, 1 mM EDTA). After one preclearing step with normal rabbit serum, the eluted Kb class I heavy chains were then reimmunoprecipitated in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 1 mM PMSF, 10 mM iodoacetamide) with the anti-p8 antiserum before gel analysis.

Bottom Line: Shortly after completion of the polypeptide chain, reactivity with KU1, KU2, and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process.Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive K(b) heavy chains.Thus, free heavy chains that arise at the cell surface are destroyed after internalization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before assembly with peptide and beta 2-microglobulin, and later, upon disintegration of the heterotrimeric complex. To characterize the structures of folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies have been produced that recognize epitopes along the H-2K(b) heavy chain which are obscured upon proper folding and subsequent assembly with beta 2-microglobulin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193-198). The K(b) heavy chain is inserted into the lumen of the endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2, and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process. Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive K(b) heavy chains. At the cell surface, a pool of free K(b) heavy chains appears after 60-120 min of chase, whose subsequent degradation, but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free heavy chains that arise at the cell surface are destroyed after internalization.

Show MeSH
Related in: MedlinePlus