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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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Appearance of unfolded class I heavy chains at the cell surface. Con A-stimulated splenocytes were pulse-labeled for 5 min and  chased at either 37° or 26°C for the times indicated. Free (Raf  HC; recognizes both Kb and Db) or KU2-reactive heavy chains (Kb alone) were immunoprecipitated from precleared lysates and resolved on SDS–PAGE  (A) or 1D–IEF (B) gels.
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Figure 5: Appearance of unfolded class I heavy chains at the cell surface. Con A-stimulated splenocytes were pulse-labeled for 5 min and chased at either 37° or 26°C for the times indicated. Free (Raf  HC; recognizes both Kb and Db) or KU2-reactive heavy chains (Kb alone) were immunoprecipitated from precleared lysates and resolved on SDS–PAGE (A) or 1D–IEF (B) gels.

Mentions: For the longer (5 min) pulse–chase experiment shown in Fig. 5, aliquots of cells (Con A-stimulated splenocytes) were spun down at each timepoint, lysed in NP-40 lysis buffer, and the postnuclear supernatant precleared once with a 1:1 mixture of normal rabbit and normal mouse serum with 50 μl S. aureus before immunoprecipitation of class I heavy chains. For the pulse– chase in Fig. 6, owing to the high background commonly observed in immunoprecipitates from RMA cell lysates, we employed the direct immunoprecipitation/reimmunoprecipitation methodology described above. In vitro transcription and translation reactions were performed as described (22).


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)

Appearance of unfolded class I heavy chains at the cell surface. Con A-stimulated splenocytes were pulse-labeled for 5 min and  chased at either 37° or 26°C for the times indicated. Free (Raf  HC; recognizes both Kb and Db) or KU2-reactive heavy chains (Kb alone) were immunoprecipitated from precleared lysates and resolved on SDS–PAGE  (A) or 1D–IEF (B) gels.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Appearance of unfolded class I heavy chains at the cell surface. Con A-stimulated splenocytes were pulse-labeled for 5 min and chased at either 37° or 26°C for the times indicated. Free (Raf  HC; recognizes both Kb and Db) or KU2-reactive heavy chains (Kb alone) were immunoprecipitated from precleared lysates and resolved on SDS–PAGE (A) or 1D–IEF (B) gels.
Mentions: For the longer (5 min) pulse–chase experiment shown in Fig. 5, aliquots of cells (Con A-stimulated splenocytes) were spun down at each timepoint, lysed in NP-40 lysis buffer, and the postnuclear supernatant precleared once with a 1:1 mixture of normal rabbit and normal mouse serum with 50 μl S. aureus before immunoprecipitation of class I heavy chains. For the pulse– chase in Fig. 6, owing to the high background commonly observed in immunoprecipitates from RMA cell lysates, we employed the direct immunoprecipitation/reimmunoprecipitation methodology described above. In vitro transcription and translation reactions were performed as described (22).

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