Transporter Associated With Antigen Processing Complex Research Articles

Tapasin Is Required for Efficient Peptide Binding to Transporter Associated with Antigen Processing

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.

Kinetic analysis of peptide binding to the TAP transport complex: evidence for structural rearrangements induced by substrate binding

The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex.

Biosynthesis of HLA-C heavy chains in melanoma cells with multiple defects in the expression of HLA-A, -B, -C molecules.

Recent investigations have shown that malignant transformation may down-regulate the expression of class I HLA molecules, β2-microglobulin (β2m) and members of the antigen-processing machinery. In the present study, we HLA-genotyped and identified at a biochemical level the three (HLA-A25, -B8, -Cw7) class I alleles expressed by the previously described [D'Urso CM et al (1992) J Clin Invest 87: 284–292] β2m-defective human melanoma FO-1 cell line and tested their ability to interact with calnexin, calreticulin and the TAP (transporter associated with antigen processing) complex. All these alleles were found to bind calnexin, but not calreticulin or the poorly expressed TAP complex, both in parental and β2m-transfected FO-1 cells, demonstrating a complex defect of class I expression in FO-1 cells. In these conditions, Cw7 heavy chains interacted with calnexin more strongly than A25 and B8, and preferentially accumulated in the endoplasmic reticulum, in both a calnexin-associated and a calnexin-free form. In addition, they could be transported to the cell surface at low levels even in the absence of β2m, without undergoing terminal glycosylation. These results establish a parallel between HLA-C and the murine Db and Ld molecules which have been found to be surface expressed and functional in β2m-defective cells. They also demonstrate distinctive features of HLA-C molecules. We propose that the accumulation of several assembly intermediates of HLA-C might favour the binding of peptide antigens not readily bound by HLA-A and -B molecules in neoplastic cells with suboptimal class I expression. © 1999 Cancer Research Campaign

Immunodeficiency due to defective antigen processing: the molecular basis for type 1 bare lymphocyte syndrome.

Immunodeficiency due to defective antigen processing: the molecular basis for type 1 bare lymphocyte syndrome.

A role for the thiol-dependent reductase ERp57 in the assembly of MHC class I molecules.

An important mammalian defence strategy against intracellular pathogens is the presentation of cytoplasmically derived short peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes. MHC class I molecules assemble in the endoplasmic reticulum (ER) with chaperones, including calnexin and calreticulin, before binding to the transporter associated with antigen processing (TAP). We show here that the thiol-dependent reductase ERp57 (also known as ER60 protease) is involved in MHC class I assembly. ERp57 co-purified with the rat TAP complex (comprising TAP1 and TAP2), and associated with MHC class I molecules at an early stage in their biosynthesis. This association was sensitive to castanospermine, which inhibits the processing of glycoproteins. Human MHC class I molecules were also found to associate with ERp57. We conclude that ERp57 is a newly identified component of the MHC class I pathway, and that it appears to interact with MHC class I molecules before they associate with TAP.

TAP- and tapasin-dependent HLA-E surface expression correlates with the binding of an MHC class I leader peptide

Background: The human major histocompatibility complex (MHC) class lb molecule HLA-E is transcribed in most tissues but little is known about its localisation within the cell. We have recently shown that HLA-E binds signal-sequence-derived peptides from human MHC class I molecules in vitro.Results: Using a newly characterised antibody recognising HLA-E, we show that HLA-E is expressed at the cell surface. We demonstrate that HLA-E surface expression is correlated with the presence of MHC class I molecules which provide suitable leader sequence peptides capable of binding to HLA-E. Further studies on the interaction of HLA-E with molecules in the endoplasmic reticulum revealed that HLA-E associates with the transporter associated with antigen processing (TAP) and calreticulin, and that HLA-E expression is TAP-dependent and tapasin-dependent. In addition, HLA-E dissociates from TAP upon binding of MHC class I leader sequence peptides.Conclusion: These experiments establish that surface expression of HLA-E is regulated by the binding of a restricted pool of peptides from the leader sequence of MHC class I molecules. The correlation between HLA-E and MHC class I surface expression might be relevant to the function of HLA-E. Our results also show that, although these HLA-E binding peptides are derived from signal sequences, they may be released back into the cytosol and subsequently translocated by the TAP complex and loaded onto HLA-E molecules.

Peptide binding and photo-crosslinking to detergent solubilized and to reconstituted transporter associated with antigen processing (TAP)

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non-radioactive crosslinking assay with a photo-activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent-solubilized, active TAP into proteoliposomes.

Major histocompatibility complex class I molecules interact with both subunits of the transporter associated with antigen processing, TAP1 and TAP2.

Prior to loading antigenic peptides, assembled major histocompatibility complex (MHC) class I molecules associate with the transporter associated with antigen processing (TAP) in a complex which also includes calreticulin and a recently described component, tapasin. The interaction of MHC class I molecules has been characterized as occurring exclusively with the TAP1 chain of the TAP heterodimer. In contrast, as described here, in the TAP-deficient human cell line T2, MHC class I molecules interact with a transfected rat TAP2 polypeptide in addition to rat TAP1. Furthermore, this interaction with TAP2 also involves calreticulin and tapasin. An association with both TAP polypeptides would presumably further enhance the efficiency of peptide loading of MHC class I molecules by allowing more than one MHC class I allele proximity to the site of peptide supply on each TAP complex.

Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast.

Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing. An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major-histocompatibility-complex class-I complexes. Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae. Expression of both genes resulted in the formation of a stable TAP heterodimer that was localized mainly in the ER. Although a minor fraction of TAP is found in the plasma membrane, TAP is unable to restore a-factor secretion in a mutant cell line that lacks the yeast mating-factor transporter Ste6. Nevertheless, in vitro studies with microsomal vesicles demonstrated that the TAP complex is fully functional in the ER membrane in terms of selective peptide binding, ATP-dependent transport, and specific inhibition by the viral protein of herpes simplex virus ICP47. This offers opportunities for topological, structural and mechanistic studies as well as genetic screenings for TAP functionality.

Peptide selection for presentation by HLA class I: a role for the human transporter associated with antigen processing?

Peptide epitopes presented by HLA class I are supplied by an elaborate system of antigen processing which subjects candidate epitopes to a process of selection according to little-understood rules. Transport of peptides from the cytosol into the endoplasmic reticulum by the TAP (transporter associated with antigen processing) complex is likely to play an important role in peptide selection for presentation. The recent development of an assay measuring substrate binding to the TAP complex has led to the identification of the major structural properties of peptides selected by the human transporter. Human TAP favors transport of peptides with structural features common to HLA class I ligands. However, TAP dislikes peptide ligands preferred by some HLA class I alleles, which may therefore frequently rely on alternative sources of peptide ligands.

Deglucosylation of N-linked glycans is an important step in the dissociation of calreticulin-class I-TAP complexes.

Recent evidence indicates that newly synthesized major histocompatibility complex (MHC) class I proteins interact with calnexin, a transmembrane endoplasmic reticulum protein specific for certain glycoproteins bearing monoglucosylated glycans. Here, we studied the association of newly synthesized class I proteins with calreticulin, a soluble calnexin-related ER protein, in murine T cells. We found that, unlike calnexin-class I interactions, calreticulin assembly with class I proteins was markedly decreased in the absence of beta 2 microglobulin expression and that calreticulin associated with a subset of class I glycoforms distinct from those assembled with calnexin but similar to those bound to TAP (transporter associated with antigen processing) proteins. Finally, these studies show that deglucosylation of N-linked glycans is important for dissociation of class I proteins from both calreticulin and TAP and that the vast majority of newly synthesized class I proteins associated with calreticulin are simultaneously assembled with TAP. The data demonstrate that calnexin and calreticulin chaperones assemble with distinct MHC class I assembly intermediates in the ER and show that glycan processing is functionally coupled to release of MHC class I proteins from peptide transport molecules.

Binding of H-2Kb-specific Peptides to TAP and Major Histocompatibility Complex Class I in Microsomes from Wild-type, TAP1, and β2-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.

Point mutations in the α2 domain of HLA-A2.1 define a functionally relevant interaction with TAP

Background: Glycoproteins encoded by the major histocompatibility complex class I region (MHC class I) present peptide antigens to cytotoxic T cells (CTLs). Peptides are delivered to the site of MHC class I assembly by the transporter associated with antigen processing (TAP), and cell lines that lack this transporter are unable to present endogenous antigens to CTLs. Although it has been shown that a fraction of newly synthesized class I molecules are in physical association with TAP, it is not known whether this interaction is functionally relevant, or where on the class I molecule the TAP binding site might be.Results C1R cells transfected with a mutant HLA-A2.1 heavy chain (HC), where threonine at position 134 in the α2 domain is changed to lysine (T134K), are unable to present endogenous antigens to CTLs. We have studied the biochemistry of this mutant in C1R cells, and found that a large pool of unstable empty class I HC–β2m (β-2 microglobulin) heterodimers exist that are rapidly transported to the cell surface. The T134K mutant seemed to bind peptide antigens and assemble with β2m as efficiently as wild-type HLA-A2.1. However, we show here that the inefficiency with which T134K presents intracellular antigen is associated with its inability to interact with the TAP heterodimer.Conclusion These experiments establish that the class I–TAP interaction is obligatory for the presentation of peptide epitopes delivered to the endoplasmic reticulum (ER) by TAP. Wild-type HLA-A2.1 molecules in TAP-deficient cells are retained in the ER, whereas T134K is rapidly released to the cell surface, but is unstable, suggesting a role for the TAP complex as an intracellular checkpoint that only affects the release of class I molecules with stably bound peptide ligands.

Requirements for Peptide Binding to the Human Transporter Associated with Antigen Processing Revealed by Peptide Scans and Complex Peptide Libraries

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

Peptide translocation by variants of the transporter associated with antigen processing.

Major histocompatibility complex (MHC) class I molecules associate with peptides that are delivered from the cytosol to the lumen of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Liver microsomes of SHR and Lewis rats, which express different alleles of TAP (cim(b) and cim(a), respectively), accumulate different sets of peptides. Use of MHC congenic rats assigned this difference to the MHC, independent of the class I products expressed. Both the cim(a) and cim(b) TAP complexes translocate peptides with a hydrophobic carboxyl terminus, but translocation of peptides with a carboxyl-terminal His, Lys, or Arg residue is unique to cim(a). Thus, the specificity of the TAP peptide translocator restricts the peptides available for antigen presentation.

Kinetics of lysozyme-substrate interactions

The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex.