Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.

The human cytomegalovirus genome encodes proteins that trigger destruction of newly synthesized major histocompatibility complex (MHC) class I molecules. The human cytomegalovirus gene US2 specifies a product capable of dislocating MHC class I molecules from the endoplasmic reticulum to the cytosol and delivering them to the proteasome. This process involves the Sec61 complex, in what appears to be a reversal of the reaction by which it translocates nascent chains into the endoplasmic reticulum.

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 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.

Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection.

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin.

Herpes simplex virus blocks intracellular transport of HLA-G in placentally derived human cells.

Spontaneous fetal loss is associated with herpes simplex virus (HSV) infection as deduced from epidemiologic data. To date, the underlying mechanisms remain to be elucidated, but an immune component is suspected. HLA-G is a class I MHC molecule selectively expressed on extravillous cytotrophoblast; this cell type does not express conventional HLA-A or -B, whereas expression of novel HLA-C-like products has been reported.

Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP.

The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane. At present, ICP47 is the only inhibitor of TAP. Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP.

A monoclonal antibody that causes the heterotrimeric G-protein G(o) to release its beta gamma subunits.

Heterotrimeric (alpha beta gamma) guanine nucleotide binding proteins (G-proteins) dissociate into their constituent subunits in the course of signal transduction. Exposure of the G-protein G(o) to the alpha(o)-specific monoclonal antibody 3E7 results in recovery of alpha(o) alone. We identified the 3E7 epitope as ERSKAIEKNL (positions 14-23) using synthetic peptides and phage display.

Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading.

Destruction of li by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complexes to the cell surface. The cysteine protease cathepsin S is highly expressed in spleen, lymphocytes, monocytes, and other class II-positive cells, and is inducible with interferon-gamma. Specific inhibition of cathepsin S in B lymphoblastoid cells prevented complete proteolysis of li, resulting in accumulation of a class II-associated 13 kDa li fragment in vivo.

The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol.

Human cytomegalovirus (HCMV) down-regulates expression of MHC class I products by selective proteolysis. A single HCMV gene, US11, which encodes an endoplasmic reticulum (ER) resident type-I transmembrane glycoprotein, is sufficient to cause this effect. In US11+cells, MHC class I molecules are core-glycosylated and therefore inserted into the ER.

Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway.

We have generated mice lacking H2-M complexes, critical facilitators of peptide loading onto major histo-compatibility complex class II molecules. Ab molecules in these mice matured into stable complexes and were efficiently expressed at the cell surface. Most carried a single peptide derived from the class II-associated invariant chain; the diverse array of peptides normally displayed by class II molecules was absent.

Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.

Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways.

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment.

Misfolded membrane proteins are rapidly degraded, often shortly after their synthesis and insertion in the endoplasmic reticulum (ER), but the exact location and mechanisms of breakdown remain unclear. We have exploited the requirement of MHC class I molecules for peptide to achieve their correct conformation: peptide can be withheld by introducing a null mutation for the MHC-encoded peptide transporter, TAP. By withholding TAP-dependent peptides, the vast majority of newly synthesized class I molecules fails to leave the endoplasmic reticulum and is degraded.

Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.

In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules.

Herpes simplex virus turns off the TAP to evade host immunity.

Many viruses have evolved mechanisms to avoid detection by the host immune system. Herpes simplex virus (HSV) expresses an immediate early protein, ICP47, which blocks presentation of viral peptides to MHC class I-restricted cells. The properties of the newly synthesized class I molecules in HSV-infected cells resemble those of cell lines deficient in the transporter associated with antigen processing (TAP) in that class I molecules are retained in the endoplasmic reticulum, and the heavy chain and beta 2-microglobulin subunits dissociate in detergent extracts but the complex can be stabilized by peptides.

Concanamycin B inhibits the expression of newly-synthesized MHC class II molecules on the cell surface.

We screened natural chemical compounds which inhibit the expression of newly-synthesized MHC class II molecules on the cell surface. IFN-gamma stimulates Colo 205 adenocarcinoma cells to induce the expression of MHC class I, MHC class II, and ICAM-1 molecules on the cell surface. We show here that concanamycin B inhibits the expression of MHC class II molecules on Colo 205 cells, whereas it does not affect the expression of MHC class I and ICAM-1 molecules.

MHC class I expression and CD8+ T cell development in TAP1/beta 2-microglobulin double mutant mice.

We have bred to homozygosity gene disruptions for the transporter associated with antigen processing 1 (TAP1) and beta 2-microglobulin (beta 2m), each of which plays a distinct role in providing class I MHC subunits. Surface expression of H-2Kb or Db on cells derived from TAP1/beta 2m -/- mice was undetectable by immunofluorescence or immunoprecipitation, unlike the situation observed for TAP1 -/- and beta 2m -/- single mutant mice. Yet, TAP1/beta 2m -/- cells were able to elicit a CD8+ cytotoxic T cell (CTL) response in mice of different H-2 haplotypes and could be killed by anti-H-2b specific CTL.

Peptide influences the folding and intracellular transport of free major histocompatibility complex class I heavy chains.

Class I major histocompatibility complex molecules require both beta 2-microglobulin (beta 2m) and peptide for efficient intracellular transport. With the exception of H-2Db and Ld, class I heavy chains have not been detectable at the surface of cells lacking beta 2m. We show that properly conformed class I heavy chains can be detected in a terminally glycosylated form indicative of cell surface expression in H-2b, H-2d, and H-2s beta 2m-/- concanavalin A (Con A)-stimulated splenocytes incubated at reduced temperature.

Increase in positive selection of CD8+ T cells in TAP1-mutant mice by human beta 2-microglobulin transgene.

Mice harboring a deletion of the gene encoding the transporter associated with antigen presentation-1 (TAP1) are impaired in providing major histocompatibility complex (MHC) class I molecules with peptides of cytosolic origin and lack stable MHC class I cell surface expression. They consequently have a strongly reduced number of CD8+ T cells. To examine whether selection of CD8+ T cells is dependent on TAP-dependent peptides, we partially restored MHC class I cell surface expression in TAP1-deficient mice by introduction of human beta 2-microglobulin.

How MHC class II molecules reach the endocytic pathway.

We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)-ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide-loaded, SDS-stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods.

How MHC class II molecules acquire peptide cargo: biosynthesis and trafficking through the endocytic pathway.

The antigen-specific receptors of T lymphocytes rely on products of the major histocompatibility complex (MHC) to recognize and engage antigen. MHC molecules display antigen on the cell surface in the form of small peptides, generated intracellularly by fragmentation of the intact protein antigen. They acquire these peptides at distinct intracellular locations: In the endoplasmic reticulum (ER), class I molecules bind peptides derived from cytosolic proteins, whereas class II molecules acquire their peptide cargo in an endocytic compartment.

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and beta 2-microglobulin mutant mice.

TAP1 -/- and beta 2-microglobulin (beta 2m) -/- mice (H-2b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8+ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8+ T cells than beta 2m -/- mice.