Craniopharyngioma in children. Surgical treatment by a transbasal anterior approach

The proper folding and assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER) is an intricate process involving a number of components. Nascent heavy chains of MHC class I molecules, translocated into the ER membrane, are rapidly glycosylated and bind the transmembrane chaperone calnexin. In humans, after dissociation from calnexin, fully oxidized MHC class I heavy chains associate with beta 2-microglobulin (beta 2m) and the soluble chaperone calreticulin. This complex interacts with another transmembrane protein, tapasin, which is believed to assist in MHC class I folding as well as in mediating the interaction between assembling MHC class I molecules and the transporter associated with antigen processing (TAP). The TAP heterodimer (TAP1-TAP2) introduces the final component of the MHC class I molecule by translocating peptides, predominately generated by the proteasome, from the cytosol into the ER where they can bind dimers of beta 2M and the MHC class I heavy chain. Recently, the thiol oxidoreductase ERp57--also known as GRP58, ERp61, ER60, Q2, HIP-70, and CPT and first misidentified as phospholipase C-alpha--has been shown to bind in conjunction with calnexin or calreticulin to a number of newly synthesized ER glycoproteins when their N-linked glycans are trimmed by glucosidases I and II. It was speculated that ERp57 is a generic component of the glycan-dependent ER quality control system. Here, we show that ERp57 is a component of the MHC class I peptide-loading complex. ERp57 might influence the folding of MHC class I molecules at a critical step in peptide loading.     

Trend in genetic medicine

The assembly of newly synthesized MHC class I molecules within the endoplasmic reticulum and their association with the transporter associated with antigen processing (TAP) is a process involving the chaperones calnexin and calreticulin. Using peptide mapping by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify a new component, we now introduce a third molecular chaperone, the thiol-dependent reductase ER-60 (ERp57/GRP58/ERp61/HIP-70/Q2), into this process. ER-60 is found in MHC class I heavy chain complexes with calnexin that are generated early during the MHC class I assembly pathway. The thiol reductase activity of ER-60 raises the possibility that ER-60 is involved in the disulfide bond formation within heavy chains. In addition, ER-60 is part of the late assembly complexes consisting of MHC class I, tapasin, TAP, calreticulin and calnexin. In a beta2-microglobulin (beta2m)-negative mouse cell line, S3, ER-60-calnexin-heavy chain complexes are shown to bind to TAP, suggesting that beta2m is not required for the association of MHC class I heavy chains with TAP.     

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells

Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.     

Indices of carbohydrate and lipid metabolism in vasopressin-replete and -deficient New Zealand genetically hypertensive rats

Intracellular antigens are continually presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic 43 kDa heavy chain and a 12 kDa soluble subunit beta 2-microglobulin (beta 2m), and which bind an 8-10 amino-acid antigenic peptide. The assembly of this trimolecular complex takes place in the lumen of the endoplasmic reticulum (ER) and almost certainly requires cofactors. Most MHC class I molecules in the ER that have not yet acquired peptide are simultaneously bound to the transporter associated with antigen processing (TAP), to the 48 kDa glycoprotein tapasin and to the lectin-like chaperone calreticulin, in a multicomponent 'loading complex'. Previous studies have shown that a mutant MHC class I molecule T134K (in which Thr134 was changed to Lys) fails to bind to TAP. Here, we show that this point mutation also disrupted, directly or indirectly, the interaction between MHC class I molecules and calreticulin. T134K molecules did not present viral antigens to T cells even though they bound peptide and beta 2m normally in vitro. They exited the ER rapidly as 'empty' MHC class I complexes, unlike empty wild-type molecules which are retained in the ER and degraded. We show here that, paradoxically, the rapid exit of empty T134K molecules from the ER was dependent on a TAP-derived supply of peptides. This implies that MHC class I assembly is a two-stage process: initial binding of suboptimal peptides is followed by peptide optimisation that depends on temporary ER retention.     

Binding of H-2Kb-specific peptides to TAP and major histocompatibility complex class I in microsomes from wild-type, TAP1, and beta2-microglobulin mutant mice

Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1(-/-) mice. In contrast, no peptide binding to beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1(-/-) microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC.beta2m heterodimers.     

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.     

Regulation of MHC class I heterodimer stability and interaction with TAP by tapasin

Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and beta 2-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP.     

Promiscuous liberation of MHC-class I-binding peptides from the C termini of membrane and soluble proteins in the secretory pathway

TAP can efficiently transport peptides up to twice as long as those bound to MHC class I molecules, suggesting a role for endoplasmic reticulum (ER) proteases in the trimming of TAP-transported peptides. To better define ER processing of antigenic peptides, we examined the capacity of TAP-deficient cells to present determinants derived from ER-targeted proteins encoded by recombinant vaccinia viruses. TAP-deficient cells failed to present antigenic peptides from internal locations in secreted proteins to MHC class I-restricted T lymphocytes. The same peptides were liberated from the C termini of a secreted protein and the lumenal domains of two membrane proteins delivered to the ER via different routes. These findings suggest that proteases in the secretory compartment can liberate C-terminal antigenic peptides from virtually any context. We propose that this activity often participates in the removal of N-terminal extensions from TAP-transported peptides, thereby creating optimally sized products for MHC class I binding. We further demonstrate that ER trimming of C termini can occur if we express an appropriate carboxypeptidase in the secretory pathway. The absence of such trimming under normal circumstances suggests that carboxypeptidase activity is generally deficient in the ER, consistent with the concordance between the specificity of TAP and MHC class I molecules for the same types of C-terminal residues.     

HLA-A*0201 presents TAP-dependent peptide epitopes to cytotoxic T lymphocytes in the absence of tapasin

Tapasin is a 48-kDa endoplasmic reticulum (ER)-resident glycoprotein that binds to the transporter associated with antigen processing (TAP) and mediates an interaction between TAP and newly synthesized MHC class I molecules. It is also essential for the proper antigen presenting function of HLA-A*0101 (HLA-A1), HLA-A*0801 (HLA-B8) and HLA-B*4402 (HLA-B4402). We show here that while tapasin is required for HLA-A*0201 (HLA-A2) molecules to bind to TAP, its absence does not block the presentation of HLA-A2-restricted TAP-dependent epitopes to cytotoxic T lymphocytes indicating that, unlike HLA-A1, HLA-B8 and HLA-B4402, HLA-A2 has access to the TAP-dependent peptide pool even in the absence of tapasin. Nevertheless, the overall efficiency with which HLA-A2 was loaded with optimal, stabilizing peptides was impaired in the cell line .220, resulting in a significant increase in the fraction of HLA-A2 molecules being released from the ER in a "peptide-receptive" state.     

MHC class I molecules compete in the endoplasmic reticulum for access to transporter associated with antigen processing

We have used the functionally distinct TAP alleles of the rat in cellular transfectants as tools to investigate how newly formed rat class I (RT1.A) molecules with distinct peptide requirements gain access to suitable peptides in the endoplasmic reticulum (ER). Normal maturation of RT1.Aa depends on the presence in the ER of peptides with C-terminal arginine, while restrictive TAP-B allelic group transporters fail to transport such peptides. In this situation, RT1.Aa is retained in the ER. We show that this retention is accompanied by accumulation of RT1.Aa in the ER, partly associated with TAP and partly free. In such cells, access to TAP of a second allelic product, RT1.Au, which does not require C-terminal arginine peptides, is competitively inhibited by the build-up of RT1.Aa. Nevertheless, RT1.Au loads and matures normally. Introduction of a permissive TAP-A allele competent to transport C-terminal arginine peptides releases RT1.Aa from the ER and restores RT1.Au interaction with TAP. Both class I alleles associate indiscriminately with permissive and restrictive TAP alleles. The data support the view that interaction with TAP is not a prerequisite for peptide loading by class I molecules, so long as suitable peptides are available in the ER. They further show that TAP association of a class I molecule depends on a competitive balance in the ER defined by the extent to which the peptide requirements of other class I molecules present are satisfied and not only by the intrinsic strength of the interaction with TAP.     

Histophysiological observations on the external auditory meatus, middle, and inner ear of the Weddell seal (Leptonychotes weddelli)

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B * 2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4 degrees C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B * 2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.     

Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells

Here, we show that bacteria induce de novo synthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system. The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the surface of recombinant Streptococcus gordonii is processed and presented on MHC class I molecules. This presentation is 10(6) times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to be presented efficiently on MHC class I molecules.     

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.     

Major histocompatibility complex class I viral antigen processing in the secretory pathway defined by the trans-Golgi network protease furin

Classical antigen presentation by major histocompatibility complex class I molecules involves cytosolic processing of endogenously synthesized antigens by proteasomes and translocation of processed peptides into the endoplasmic reticulum (ER) by transporters associated with antigen presentation (TAP). Alternative pathways for processing of endogenous antigens, generally involving the ER, have been suggested but not fully proved. We analyzed the potential for class I presentation of proteolytic maturation of secretory antigens in the exocytic pathway. We found that hepatitis B (HB) virus secretory core protein HBe can efficiently deliver COOH-terminally located antigenic peptides for endogenous class I loading in the absence of TAP. Antigen presentation to specific cytotoxic T lymphocytes correlates with protein maturation at the COOH terminus, since modification of maturation and transport of HBe through the secretory pathway alters antigen presentation. Both maturation and a necessary processing step occur in the Golgi or post-Golgi compartment. Antigen presentation is independent of proteasome activity, but inhibitors of the trans-Golgi network resident protease furin inhibit both HBe maturation and antigen presentation. These results define a new antigen processing pathway located in the secretory route, with a central role for proteolytic maturation mediated by the subtilisin protease family member furin as an efficient source for antigen presentation.     

MR imaging of symptomatic osteochondromas with pathological correlation

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules depends on translocation of cytosolic peptides into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Peptide transport by TAP is thought to include at least two steps: initial binding of peptide to TAP, and its subsequent translocation requiring ATP hydrolysis. These events can be monitored in peptide binding and transport assays. Previous studies have shown that the efficiency of peptide transport by human, mouse and rat transporters varies according to the C-terminals of peptide substrates in an allele and species-specific manner. However, it has not been clear during which step of peptide interaction with TAP selection occurs. We used an assay monitoring the peptide binding step to study the binding affinity of a library of 199 peptides for human TAP and the two major allelic rat TAP complexes. We observed a dominant influence of the C-terminus on peptide binding affinity for all transporters, and highly restrictive selection of peptides with aliphatic and aromatic C-terminals by rat TAP1/TAP2u complexes. The selectivity of peptide binding to rat TAP complexes is in full accordance with published data on selective peptide transport and on control of antigen presentation by rat TAP. These results strongly suggest that (i) peptide selection by TAP occurs exclusively in the initial binding step; (ii) all factors involved in peptide selection by TAP are present in insect cells.     

Analysis of posterior plagiocephaly: deformational versus synostotic

Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcgammaR), which mediate internalization of antigen-IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II-restricted antigen presentation. We now show that FcgammaRs have two additional specific attributes in murine DCs: the induction of DC maturation and the promotion of efficient MHC class I-restricted presentation of peptides from exogenous, IgG-complexed antigens. Both FcgammaR functions require the FcgammaR-associated gamma chain. FcgammaR-mediated MHC class I-restricted antigen presentation is extremely sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules, ICs should efficiently sensitize DCs for priming of both CD4(+) helper and CD8(+) cytotoxic T lymphocytes in vivo.     

A topographic study on the distribution of cisplatin in xenografted tumors on nude mice

The influence of the TAP complex on T-cell allorecognition of MHC class II molecules was examined using human B-cell lines that have mutations in the TAP 1 or 2 genes. The TAP mutations led to the loss of allorecognition for two of 28 anti- HLA-DR T-cell clones. Restoration of TAP expression by transfection of a TAP 2 cDNA clone led to recovery of the alloresponse for both clones. These results could be explained in two ways. First, TAP dependence could reflect specificity for a peptide derived from an MHC class I molecule that is less efficiently generated by the endocytic pathway in the TAP-deficient stimulator cells owing to reduction in surface class I expression. The proliferative responses of these clones to the TAP-deficient stimulator cells was not restored by rescue of cell-surface expression of class I molecules by low temperature culture or by the addition of class I-binding peptides. These data therefore favor the alternative explanation that class II loading by some peptides is TAP dependent. Circumstances that lead to the amplification of this minority pathway of endogenous presentation by class II MHC molecules may have the potential to interrupt self-tolerance.     

Nonclassical behavior of the mouse CD1 class I-like molecule

The mouse CD1 (mCD1) molecule is a class I-like molecule that is encoded outside of the MHC. We show here that mCD1 shares several properties with Ag-presenting class I molecules, including a requirement for beta2-microglobulin for stable cell-surface expression in T lymphocyte transfectants and thymocytes. mCD1 is also capable of binding to mouse CD8alphabeta heterodimers participating in the activation of CD8+ T cells in a manner similar to classical class I molecules. However, mCD1 surface expression is not decreased at high temperatures in cells that lack the transporter associated with Ag processing (TAP), including both RMA-S and Drosophila melanogaster cells. The data indicate that mCD1 does not require TAP to be expressed in a stable fashion at the cell surface. We speculate that the ability of mCD1 to reach the cell surface in transporter-deficient cells may reflect its ability to present a distinct set of ligands. The properties of mCD1 described here can account, in part, for the selection of the diverse populations of T cells that are known to be mCD1 reactive.