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

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

R P Machold et al. J Exp Med. 1996.

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.

PubMed Disclaimer

Figures

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.

Figure 3

Figure 4

Effects of redox potential and calnexin association on the folding and assembly of the Kb molecule. RMA cells were pulsed for 1 min and chased for the times indicated. Nascent heavy chains were directly immunoprecipitated from NP-40 detergent lysates as in Fig. 3 with the antibodies indicated in each panel. The reducing agent DTT (B) was added to cells at 5 mM 5 min prior to labeling, while the glucosidase inhibitor 7-O-dec (C ) was added to cells at 2 mM during starvation (45 min prior to labeling).

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.

Figure 6

Breakdown of unfolded Kb heavy chains at the cell surface. (A) RMA cells were starved for 45 min in the absence or presence of the vacuolar H+-ATPase inhibitor Con B, pulse-labeled for 5 min, and then chased for the times indicated. Class I heavy chains were directly immunoprecipitated from NP-40 detergent lysates (no initial preclear) with the antibodies indicated in each panel, and reimmunoprecipitated with the p8 antiserum before SDS–PAGE. (B) Phosphoimager quantitation of the band densities in A. The data sets for each pulse–chase were normalized around the 60 min chase point densities.

Figure 6

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.

Figure 1

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).

Figure 2

References

1. Bjorkman PJ, Parham P. Structure, function, and diversity of class I major histocompatibility complex molecules. Annu Rev Biochem. 1990;59:253–288. - PubMed
1. Yewdell JW, Bennink JR. Cell biology of antigen processing and presentation to MHC class I molecule– restricted T lymphocytes. Adv Immunol. 1992;52:1–123. - PubMed
1. Williams DB, Watts TH. Molecular chaperones in antigen presentation. Curr Opin Immunol. 1995;7:77–84. - PubMed
1. Vassilakos A, Cohen-Doyle MF, Peterson PA, Jackson MR, Williams DB. The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. EMBO (Eur Mol Biol Organ) J. 1996;15:1495–1506. - PMC - PubMed
1. Chen W, Helenius J, Braakman I, Helenius A. Cotranslational folding and calnexin binding during glycoprotein synthesis. Proc Natl Acad Sci USA. 1995;92:6229–6233. - PMC - PubMed

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