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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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Epitope mapping of the monoclonal antibodies KU1, KU2,  and KU4. (A) Three representative phage insert sequences selected by  each antibody are shown, with translation, above the Kb sequence that is  predicted to contain the epitope. Residues in the phage sequences that  match the actual Kb sequence are shown in capital letters. For comparison, sequences of other class I heavy chains are shown below each predicted epitope. (B) Ribbon diagrams of the properly conformed Kb heavy  chain, highlighting in grey the locations of the predicted epitopes.
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Figure 1: Epitope mapping of the monoclonal antibodies KU1, KU2, and KU4. (A) Three representative phage insert sequences selected by each antibody are shown, with translation, above the Kb sequence that is predicted to contain the epitope. Residues in the phage sequences that match the actual Kb sequence are shown in capital letters. For comparison, sequences of other class I heavy chains are shown below each predicted epitope. (B) Ribbon diagrams of the properly conformed Kb heavy chain, highlighting in grey the locations of the predicted epitopes.

Mentions: With the aim of characterizing partially folded or assembled forms of the Kb class I heavy chain, we raised three mAbs against denatured Kb molecules: KU1, KU2, and KU4. The immunogen was injected as urea-solubilized inclusion bodies obtained from E. coli, and presumably contained little coherent structure. Therefore, we expected that antibodies from this immunization would recognize unstructured epitopes along the Kb polypeptide chain. Thus, we employed an M13-based phage display library (19) encoding a random 10 amino acid insert to map the epitopes for each mAb. Three representative inserts are shown for each antibody (Fig. 1 A), although we sequenced at least 10 clones from each pool of selected phage to determine the anchor residues for each epitope. Beneath each consensus sequence is shown the corresponding Kb sequence and the sequences of other class I heavy chains in the same region (20). The location of each epitope could be established without ambiguity, and their projection onto the fully assembled Kb molecule is shown in Fig. 1 B (21). The epitope recognized by KU1 (residues 49–54) is normally folded into a 310 helix that immediately precedes the long α helix in the α1 domain which forms one side of the peptide-binding groove. The involvement of four out of five consecutive residues in generation of the KU1 epitope argues in favor of recognition of this stretch in nonhelical configuration and, conversely, folding of this region should conceal the KU1 epitope. KU2 binds to an epitope that spans part of the second β strand and connecting loop sequence in the α1 domain (residues 23–30); this region contains residues that directly contact β2m (Y27, E32). The KU4 epitope maps to the first β strand in the α3 domain (residues 193–198) and is adjacent to residues that contact β2m (R202, W204) as well as one of the cysteines (C203) that participates in the α3 disulfide bond. Note that the residues recognized by each antibody, as identified by phage display, are buried or rearranged upon folding of the heavy chain and assembly with β2m.


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)

Epitope mapping of the monoclonal antibodies KU1, KU2,  and KU4. (A) Three representative phage insert sequences selected by  each antibody are shown, with translation, above the Kb sequence that is  predicted to contain the epitope. Residues in the phage sequences that  match the actual Kb sequence are shown in capital letters. For comparison, sequences of other class I heavy chains are shown below each predicted epitope. (B) Ribbon diagrams of the properly conformed Kb heavy  chain, highlighting in grey the locations of the predicted epitopes.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Epitope mapping of the monoclonal antibodies KU1, KU2, and KU4. (A) Three representative phage insert sequences selected by each antibody are shown, with translation, above the Kb sequence that is predicted to contain the epitope. Residues in the phage sequences that match the actual Kb sequence are shown in capital letters. For comparison, sequences of other class I heavy chains are shown below each predicted epitope. (B) Ribbon diagrams of the properly conformed Kb heavy chain, highlighting in grey the locations of the predicted epitopes.
Mentions: With the aim of characterizing partially folded or assembled forms of the Kb class I heavy chain, we raised three mAbs against denatured Kb molecules: KU1, KU2, and KU4. The immunogen was injected as urea-solubilized inclusion bodies obtained from E. coli, and presumably contained little coherent structure. Therefore, we expected that antibodies from this immunization would recognize unstructured epitopes along the Kb polypeptide chain. Thus, we employed an M13-based phage display library (19) encoding a random 10 amino acid insert to map the epitopes for each mAb. Three representative inserts are shown for each antibody (Fig. 1 A), although we sequenced at least 10 clones from each pool of selected phage to determine the anchor residues for each epitope. Beneath each consensus sequence is shown the corresponding Kb sequence and the sequences of other class I heavy chains in the same region (20). The location of each epitope could be established without ambiguity, and their projection onto the fully assembled Kb molecule is shown in Fig. 1 B (21). The epitope recognized by KU1 (residues 49–54) is normally folded into a 310 helix that immediately precedes the long α helix in the α1 domain which forms one side of the peptide-binding groove. The involvement of four out of five consecutive residues in generation of the KU1 epitope argues in favor of recognition of this stretch in nonhelical configuration and, conversely, folding of this region should conceal the KU1 epitope. KU2 binds to an epitope that spans part of the second β strand and connecting loop sequence in the α1 domain (residues 23–30); this region contains residues that directly contact β2m (Y27, E32). The KU4 epitope maps to the first β strand in the α3 domain (residues 193–198) and is adjacent to residues that contact β2m (R202, W204) as well as one of the cysteines (C203) that participates in the α3 disulfide bond. Note that the residues recognized by each antibody, as identified by phage display, are buried or rearranged upon folding of the heavy chain and assembly with β2m.

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