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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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Western blot analysis of diverse class I material. Splenocyte  extracts from H-2 b, d, k, and u haplotype mice were resolved on SDS– PAGE or 1D–IEF gels and blotted with either RafHC (A) or KU2 (B).
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Figure 2: Western blot analysis of diverse class I material. Splenocyte extracts from H-2 b, d, k, and u haplotype mice were resolved on SDS– PAGE or 1D–IEF gels and blotted with either RafHC (A) or KU2 (B).

Mentions: The results of the epitope mapping described above and sequence comparisons for class I heavy chains in that region suggested that KU1 and KU2 would recognize Kb exclusively, and that KU4 would recognize both Kb and Kk molecules. To probe directly the specificity of the antibodies, Western blots were performed on splenocyte extracts prepared from mice of b, d, k, and u haplotype resolved on SDS-PAGE and 1D–IEF gels (Fig. 2). As a control, we blotted with the rabbit anti-free heavy serum (Raf HC), which recognizes most murine class I heavy chains (13). As anticipated, KU2 recognized Kb heavy chains exclusively (Fig. 2 B), whereas Raf HC decorated all of the class I material present (Fig. 2 A). Neither KU1 nor KU4 blotted as efficiently as KU2, although the specificity for each was as expected based on the epitope mapping (data not shown). Further confirmation of the predicted epitopes for the antibodies was obtained from immunoprecipitations performed on Kb COOH-terminal truncation fragments translated in vitro (KU1, KU2, and KU4 all react with translation products containing Kb residues 1–204; only KU1 and KU2 react with fragments containing residues 1–74; data not shown).


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)

Western blot analysis of diverse class I material. Splenocyte  extracts from H-2 b, d, k, and u haplotype mice were resolved on SDS– PAGE or 1D–IEF gels and blotted with either RafHC (A) or KU2 (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Western blot analysis of diverse class I material. Splenocyte extracts from H-2 b, d, k, and u haplotype mice were resolved on SDS– PAGE or 1D–IEF gels and blotted with either RafHC (A) or KU2 (B).
Mentions: The results of the epitope mapping described above and sequence comparisons for class I heavy chains in that region suggested that KU1 and KU2 would recognize Kb exclusively, and that KU4 would recognize both Kb and Kk molecules. To probe directly the specificity of the antibodies, Western blots were performed on splenocyte extracts prepared from mice of b, d, k, and u haplotype resolved on SDS-PAGE and 1D–IEF gels (Fig. 2). As a control, we blotted with the rabbit anti-free heavy serum (Raf HC), which recognizes most murine class I heavy chains (13). As anticipated, KU2 recognized Kb heavy chains exclusively (Fig. 2 B), whereas Raf HC decorated all of the class I material present (Fig. 2 A). Neither KU1 nor KU4 blotted as efficiently as KU2, although the specificity for each was as expected based on the epitope mapping (data not shown). Further confirmation of the predicted epitopes for the antibodies was obtained from immunoprecipitations performed on Kb COOH-terminal truncation fragments translated in vitro (KU1, KU2, and KU4 all react with translation products containing Kb residues 1–204; only KU1 and KU2 react with fragments containing residues 1–74; data not shown).

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