Intermediates in the Assembly and Degradation of Class I Major Histocompatibility Complex (MHC) Molecules Probed with Free Heavy Chain–specific Monoclonal Antibodies

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 β2-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-2Kb heavy chain which are obscured upon proper folding and subsequent assembly with β2-microglobulin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193-198). The Kb 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 Kb heavy chains. At the cell surface, a pool of free Kb 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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Characterization of a Murine Cytomegalovirus Class I Major Histocompatibility Complex (MHC) Homolog: Comparison to MHC Molecules and to the Human Cytomegalovirus MHC Homolog

Journal of Virology ◽

10.1128/jvi.72.1.460-466.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 460-466 ◽

Cited By ~ 44

Author(s):

Tara L. Chapman ◽

Pamela J. Bjorkman

Keyword(s):

Major Histocompatibility Complex ◽

Heavy Chain ◽

Class I ◽

Major Histocompatibility ◽

Class I Mhc ◽

Endogenous Peptides ◽

Β2 Microglobulin ◽

Mhc Molecules ◽

Histocompatibility Complex ◽

Infected Cells

ABSTRACT Both human and murine cytomegaloviruses (HCMV and MCMV) down-regulate expression of conventional class I major histocompatibility complex (MHC) molecules at the surfaces of infected cells. This allows the infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to natural killer cells, which lyse cells that lack class I molecules. Both HCMV and MCMV encode class I MHC heavy-chain homologs that may function in immune response evasion. We previously showed that a soluble form of the HCMV class I homolog (UL18) expressed in Chinese hamster ovary cells binds the class I MHC light-chain β2-microglobulin and a mixture of endogenous peptides (M. L. Fahnestock, J. L. Johnson, R. M. R. Feldman, J. M. Neveu, W. S. Lane, and P. J. Bjorkman, Immunity 3:583–590, 1995). Consistent with this observation, sequence comparisons suggest that UL18 contains the well-characterized groove that serves as the binding site in MHC molecules for peptides derived from endogenous and foreign proteins. By contrast, the MCMV homolog (m144) contains a substantial deletion within the counterpart of its α2 domain and might not be expected to contain a groove capable of binding peptides. We have now expressed a soluble version of m144 and verified that it forms a heavy chain–β2-microglobulin complex. By contrast to UL18 and classical class I MHC molecules, m144 does not associate with endogenous peptides yet is thermally stable. These results suggest that UL18 and m144 differ structurally and might therefore serve different functions for their respective viruses.

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Dissociation of β2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface

Cell ◽

10.1016/0092-8674(91)90093-e ◽

1991 ◽

Vol 65 (4) ◽

pp. 611-620 ◽

Cited By ~ 80

Author(s):

Kenneth L. Rock ◽

Sandra Gamble ◽

Lisa Rothstein ◽

Colette Gramm ◽

Baruj Benacerraf

Keyword(s):

Cell Surface ◽

Class I ◽

Class I Mhc ◽

Β2 Microglobulin ◽

Heavy Chains

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The Pathway of Us11-Dependent Degradation of Mhc Class I Heavy Chains Involves a Ubiquitin-Conjugated Intermediate

The Journal of Cell Biology ◽

10.1083/jcb.147.1.45 ◽

1999 ◽

Vol 147 (1) ◽

pp. 45-58 ◽

Cited By ~ 125

Author(s):

Caroline E. Shamu ◽

Craig M. Story ◽

Tom A. Rapoport ◽

Hidde L. Ploegh

Keyword(s):

Mhc Class I ◽

Heavy Chain ◽

Control Process ◽

Degradation Pathway ◽

Cell System ◽

Class I ◽

Permeabilized Cell ◽

Intact Cells ◽

Heavy Chains ◽

Quality Control Process

The human cytomegalovirus protein, US11, initiates the destruction of MHC class I heavy chains by targeting them for dislocation from the ER to the cytosol and subsequent degradation by the proteasome. We report the development of a permeabilized cell system that recapitulates US11-dependent degradation of class I heavy chains. We have used this system, in combination with experiments in intact cells, to identify and order intermediates in the US11-dependent degradation pathway. We find that heavy chains are ubiquitinated before they are degraded. Ubiquitination of the cytosolic tail of heavy chain is not required for its dislocation and degradation, suggesting that ubiquitination occurs after at least part of the heavy chain has been dislocated from the ER. Thus, ubiquitination of the heavy chain does not appear to be the signal to start dislocation. Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane. Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.

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Abrogation of cell surface expression of human class I transplantation antigens by an adenovirus protein in Xenopus laevis oocytes.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.540 ◽

1985 ◽

Vol 101 (2) ◽

pp. 540-547 ◽

Cited By ~ 16

Author(s):

L Severinsson ◽

P A Peterson

Keyword(s):

Cell Surface ◽

Heavy Chain ◽

Viral Protein ◽

Surface Expression ◽

Class I ◽

Cell Surface Expression ◽

Xenopus Laevis Oocytes ◽

Human Class ◽

Heavy Chains ◽

Transplantation Antigens

Class I transplantation antigens form complexes with a virus protein encoded in the early region E3 of the adenovirus-2 genome. The interaction between this viral glycoprotein, E19, and nascent human class I antigens has been examined by microinjecting purified mRNA into Xenopus laevis oocytes. Both E19 and the two class I antigen subunits, the heavy chain and beta 2-microglobulin (beta 2M), were efficiently translated. The heavy chains did not become terminally glycosylated, as monitored by endoglycosidase H digestion, and were not expressed on the oocyte surface unless they were associated with beta 2M. The E19 protein did not become terminally glycosylated, and we failed to detect this viral protein on the surface of the oocytes. Co-translation of heavy chain and E19 mRNA demonstrated that the two proteins associate intracellularly. However, neither protein appeared to be transported to the trans-Golgi compartment. Similar observations were made in adenovirus-infected HeLa cells. Heavy chains bound to beta 2M became terminally glycosylated in oocytes in the presence of low concentrations of E19. At high concentrations of the viral protein, no carbohydrate modifications and no cell surface expression of class I antigens were apparent. Thus, beta 2M and E19 have opposite effects on the intracellular transport of the heavy chains. These data suggest that adenovirus-2 may impede the cell surface expression of class I antigens to escape immune surveillance.

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Serologic cross-reactivity between Class I MHC molecules and an H-2-linked differentiation antigen as detected by monoclonal antibodies.

Journal of Experimental Medicine ◽

10.1084/jem.159.1.21 ◽

1984 ◽

Vol 159 (1) ◽

pp. 21-40 ◽

Cited By ~ 44

Author(s):

S O Sharrow ◽

L Flaherty ◽

D H Sachs

Keyword(s):

Monoclonal Antibodies ◽

Dna Sequences ◽

Bone Marrow Cells ◽

Tissue Expression ◽

Cross Reactivity ◽

Class I ◽

Differentiation Antigen ◽

Class I Mhc ◽

Mhc Molecules ◽

The Right

Analysis of anti-Class I major histocompatibility complex (MHC) monoclonal antibodies by immunofluorescence and flow microfluorometry demonstrated an unexpected cross-reactivity. Two of fifteen antibodies examined (20-8-4, anti-Kb,Kd,r,s and 34-1-2, antiKd,Dd,Kb,r,s,q,p) were observed to detect an antigen determined by gene(s) mapping to the right of H-2D. Two-color immunofluorescence analysis demonstrated that this antigen, unlike classical H-2K and D antigens, was expressed in high amounts on peripheral T cells, but only weakly on Ia-positive cells and on small subpopulations of thymus and bone marrow cells. Mapping, absorption, blocking, and tissue distribution studies suggested that the cross-reactive antigen is Qa-like, but distinct from previously described Qa antigens. Thus, these data demonstrate serological cross-reactivity between a Class I MHC antigen and a differentiation antigen determined by genes linked to H-2. It seems likely that the gene responsible for this new antigen is one of the numerous Class I-like sequences detected by DNA hybridization analyses, but previously undefined in terms of tissue expression. These data suggest that many of these DNA sequences may be expressed in specific tissues and that cross-reactions of anti-Class I MAbs may provide useful probes for studying the products of such homologous genes.

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Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells

Journal of Cell Science ◽

10.1242/jcs.107.7.2005 ◽

1994 ◽

Vol 107 (7) ◽

pp. 2005-2020 ◽

Cited By ~ 1

Author(s):

F. Garcia-del Portillo ◽

M.G. Pucciarelli ◽

W.A. Jefferies ◽

B.B. Finlay

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Epithelial Cells ◽

Cell Surface ◽

Heavy Chain ◽

Surface Proteins ◽

Class I ◽

Plasma Membrane Proteins ◽

Class I Mhc ◽

Host Plasma Membrane

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.

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