Integrated modeling of the major events in the MHC class I antigen processing pathway

Rational design of epitope-driven vaccines is a key goal of immunoinformatics. Typically, candidate selection relies on the prediction of MHC-peptide binding only, as this is known to be the most selective step in the MHC class I antigen processing pathway. However, proteasomal cleavage and transport by the transporter associated with antigen processing (TAP) are essential steps in antigen processing as well. While prediction methods exist for the individual steps, no method has yet offered an integrated prediction of all three major processing events. Here we present WAPP, a method combining prediction of proteasomal cleavage, TAP transport, and MHC binding into a single prediction system. The proteasomal cleavage site prediction employs a new matrix-based method that is based on experimentally verified proteasomal cleavage sites. Support vector regression is used for predicting peptides transported by TAP. MHC binding is the last step in the antigen processing pathway and was predicted using a support vector machine method, SVMHC. The individual methods are combined in a filtering approach mimicking the natural processing pathway. WAPP thus predicts peptides that are cleaved by the proteasome at the C terminus, transported by TAP, and show significant affinity to MHC class I molecules. This results in a decrease in false positive rates compared to MHC binding prediction alone. Compared to prediction of MHC binding only, we report an increased overall accuracy and a lower rate of false positive predictions for the HLA-A*0201, HLA-B*2705, HLA-A*01, and HLA-A*03 alleles using WAPP. The method is available online through our prediction server at http://www-bs.informatik.uni-tuebingen.de/WAPP     

A neural network model approach to the study of human TAP transporter

We used an artificial neural network (ANN) computer model to study peptide binding to the human transporter associated with antigen processing (TAP). After validation, an ANN model of TAP-peptide binding was used to mine a database of HLA-binding peptides to elucidate patterns of TAP binding. The affinity of HLA-binding peptides for TAP was found to differ according to the HLA supertype concerned: HLA-B27, -A3 or -A24 binding peptides had high, whereas HLA-A2, -B7 or -B8 binding peptides had low affinity for TAP. These results support the idea that TAP and particular HLA molecules may have co-evolved for efficient peptide processing and presentation. The strong similarity between the sets of peptides bound by TAP or HLA-B27 suggests functional co-evolution whereas the lack of a relationship between the sets of peptides bound by TAP or HLA-A2 is against these particular molecules having co-evolved. In support of these conclusions, the affinities of HLA-A2 and HLA-B7 binding peptides for TAP show similar distributions to that of randomly generated peptides. On the basis of these results we propose that HLA alleles constitute two separate classes: those that are TAP-efficient for peptide loading (HLA-B27, -A3 and -A24) and those that are TAP-inefficient (HLA-A2, -B7 and -B8). Computer modelling can be used to complement laboratory experiments and thereby speed up knowledge discovery in biology. In particular, we provide evidence that large-scale experiments can be avoided by combining initial experimental data with limited laboratory experiments sufficient to develop and validate appropriate computer models. These models can then be used to perform large-scale simulated experiments the results of which can then be validated by further small-scale laboratory experiments.     

NetCTLpan: pan-specific MHC class I pathway epitope predictions

Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I ligands and cytotoxic T lymphocyte (CTL) epitopes. It has been reported that MHC molecules are differentially dependent on TAP transport and proteasomal cleavage. Here, we did not find any consistent signs of such MHC dependencies, and the NetCTLpan method is implemented with fixed weights for proteasomal cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying MHC class I epitopes. Using the NetCTLpan method, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively, when compared to the NetMHCpan and NetCTL methods. The method and benchmark datasets are available at .     

Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.     

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.     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.     

主要组织相容性复合体I类分子抗原肽

被主要组织相容性复合体(MHC)I类分子呈递在细胞表面的抗原肽大部分来源于细胞内新合成蛋白质的降解产物,抗原肽直接体现细胞内功能蛋白质的部分变化,蛋白酶体、氨肽酶和抗原转运体(TAP)参与调控抗原肽的生成。在MHC的组装、折叠过程中,抗原肽促进各亚基的结合和折叠进程;而在起始细胞的免疫应答过程中,抗原肽不仅诱导T细胞抗原受体的特异结合,更为重要的是延长MHC同T细胞抗原受体特异结合的作用时间。     

抗原处理相关转运体蛋白的研究进展

抗原处理相关转运体(transporter associated with antigen processing,TAP)蛋白在抗原提呈途径中发挥重要作用,它负责将内源性抗原从胞浆运送到内质网(endoplasmic reticulum,ER),以便主要组织相容性复合体(major histocompatibility complex,MHC)I结合多肽。TAP属于ATP结合盒(ATP-binding cassette,ABC)转运蛋白超家族B族,是由TAP1和TAP2两个亚基构成的异二聚体蛋白,其每个亚基各含有一个亲水的核酸结合区和一个疏水的跨膜结构域,并具有促进肽段转运的结构域。TAP参与MHC I类分子的组装,并在人获得性免疫系统中起着至关重要的作用。TAP基因具有多态性,因而增加了个体对疾病的易感性。TAP基因的突变及其调节机制的缺陷都可以导致其活性和表达下调,从而影响病毒性感染和肿瘤等疾病的发生。     

Crystal structures of two rat MHC class Ia (RT1-A) molecules that are associated differentially with peptide transporter alleles TAP-A and TAP-B

Antigenic peptides are loaded onto class I MHC molecules in the endoplasmic reticulum (ER) by a complex consisting of the MHC class I heavy chain, beta(2)-microglobulin, calreticulin, tapasin, Erp57 (ER60) and the transporter associated with antigen processing (TAP). While most mammalian species transport these peptides into the ER via a single allele of TAP, rats have evolved different TAPs, TAP-A and TAP-B, that are present in different inbred strains. Each TAP delivers a different spectrum of peptides and is associated genetically with distinct subsets of MHC class Ia alleles, but the molecular basis for the conservation (or co-evolution) of the two transporter alleles is unknown. We have determined the crystal structures of a representative of each MHC subset, viz RT1-A(a) and RT1-A1(c), in association with high-affinity nonamer peptides. The structures reveal how the chemical properties of the two different rat MHC F-pockets match those of the corresponding C termini of the peptides, corroborating biochemical data on the rates of peptide-MHC complex assembly. An unusual sequence in RT1-A1(c) leads to a major deviation from the highly conserved beta(3)/alpha(1) loop (residues 40-59) conformation in mouse and human MHC class I structures. This loop change contributes to profound changes in the shape of the A-pocket in the peptide-binding groove and may explain the function of RT1-A1(c) as an inhibitory natural killer cell ligand.     

Function of the transport complex TAP in cellular immune recognition

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility complex (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. The MHC-encoded ABC transporter works in concert with the proteasome and MHC class I molecules for the antigen presentation on the cell surface for T cell recognition. TAP forms a heterodimer where each subunit consists of a hydrophilic nucleotide binding domain and a hydrophobic transmembrane domain. The transport mechanism is a multistep process composed of an ATP-independent peptide association step which induces a structural reorganization of the transport complex that may trigger the ATP-driven transport of the peptide into the endoplasmic reticulum lumen. By using combinatorial peptide libraries, the substrate selectivity and the recognition principle of TAP have been elucidated. TAP maximizes the degree of substrate diversity in combination with high substrate affinity. This ABC transporter is also unique as it is closely associated with chaperone-like proteins involved in bonding of the substrate onto MHC molecules. Most interestingly, virus-infected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function.     

Distinct functions of the ATP binding cassettes of transporters associated with antigen processing: a mutational analysis of Walker A and B sequences

The transporters associated with antigen processing (TAP1/TAP2) provide peptides to MHC class I molecules in the endoplasmic reticulum. Like other ATP-binding cassette proteins, TAP uses ATP hydrolysis to power transport. We have studied peptide binding to as well as translocation by TAP proteins with mutations in the Walker A and B sequences that are known to mediate ATP binding and hydrolysis. We show that a mutation in the TAP1 Walker B sequence reported to abrogate class I expression by a lung tumor does not affect ATP binding affinity, suggesting a defect restricted to ATP hydrolysis. This mutation reduces peptide transport by only 50%, suggesting that TAP function can be highly limiting for antigen presentation in non-lymphoid cells. Single substitutions in Walker A sequences (TAP1K544A, TAP2K509A), or their complete replacements, abrogate nucleotide binding to each subunit. Although all of these mutations abrogate peptide transport, they reveal distinct roles for nucleotide binding to the two transporter subunits in TAP folding and in regulation of peptide substrate affinity, respectively. Alteration of the TAP1 Walker A motif can have strong effects on TAP1 and thereby TAP complex folding. However, TAP1 Walker A mutations compatible with correct folding do not affect peptide binding. In contrast, abrogation of the TAP2 nucleotide binding capacity has little or no effect on TAP folding but eliminates peptide binding to TAP at 37 degrees C in the presence of nucleotides. Thus, nucleotide binding to TAP2 but not to TAP1 is a prerequisite for peptide binding to TAP. Based on these results, we propose a model in which nucleotide and peptide release from TAP are coupled and followed by ATP binding to TAP2, which induces high peptide affinity and initiates the transport cycle.     

Nucleotide binding by TAP mediates association with peptide and release of assembled MHC class I molecules

Background: Newly synthesised peptide-receptive major histocompatibility complex (MHC) class I molecules form a transient loading complex in the endoplasmic reticulum with the transporter associated with antigen processing (TAP) and a set of accessory proteins. Binding of peptide to the MHC class I molecule is necessary for dissociation of the MHC class I molecule from the complex with TAP, but other components of the complex might also be involved. To investigate the role of TAP in this process, mutations that block nucleotide binding were introduced into the ATP-binding site of TAP.Results: Mutant TAP formed apparently normal loading complexes with MHC class I molecules and accessory components, but had no nucleotide-binding or peptide-transport activity. Nevertheless, whereas wild-type loading complexes in detergent lysates could be dissociated by addition of peptides that bind MHC class I molecules, mutant complexes could not be dissociated in this way. Depletion of nucleotide diphosphates or triphosphates from wild-type lysates blocked peptide-mediated dissociation of MHC class I molecules, which could be reversed by readdition of nucleotide diphosphates or triphosphates. Complexes between mutant TAP and MHC class I molecules remained associated in vivo until they were degraded. Disruption of nucleotide binding also eliminated TAP's peptide-binding activity.Conclusions: Peptide-mediated dissociation of the MHC class I molecule from the loading complex depends on conformational signals arising from TAP. Integrity of the nucleotide-binding site is required not only for transmission of this conformational signal to the loading complex, but also for binding of peptide to TAP. Thus, the dynamic activity of the loading complex is synchronised with the nucleotide-mediated peptide-binding and transport cycle of TAP.     

Traffic of proteins and peptides across membranes for immunosurveillance by CD8(+) T lymphocytes: a topological challenge

Cytotoxic CD8(+) T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen-derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8(+) T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full-length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8(+) T lymphocytes and highlights the need to molecularly identify the transporters involved.     

Host immune system strikes back

Autophagy-mediated major histocompatibility complex (MHC) class I presentation can follow either the conventional MHC class I pathway or a recently described vacuolar pathway. In the vacuolar pathway, protein degradation is effected by lysosomal proteases, peptide exchange takes place with recirculating MHC complexes and the newly formed peptide-MHC complexes reach the cell surface by the endocytic pathway. This pathway is independent of the proteasome and the transporter associated with antigen processing (TAP) complex, but generates the same, or a similar, epitope as that from the conventional MHC class I pathway. Here, we discuss different mechanisms by which autophagy mediates MHC class I-restricted antigen presentation, which is crucial to its role in the control of intracellular pathogens.     

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.     

Disruption of the association of 73 kDa heat shock cognate protein with transporters associated with antigen processing (TAP) decreases TAP-dependent translocation of antigenic peptides into the endoplasmic reticulum

Major histocompatibility complex class I-bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10 mM ATP, indicating that the ADP-binding form of HSC73 might be associated with TAP. We found that HSC73-binding immunosuppressant, MeDSG disrupted the HSC73-TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP-mediated ER translocation was examined using two homologous model peptides, NGT-Bw4 and NGT-Bw6, which had distinct binding affinity to HSC73. Although high-affinity peptide NGT-Bw4 was translocated by TAP, low-affinity peptide NGT-Bw6 was not. The TAP-dependent translocation of NGT-Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA)-A31-restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73-TAP association resulted in inhibition of TAP-dependent translocation of HSC73-bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I-restricted presentation of HSC73-bound antigenic peptides.     

The Intracellular Antigen Transport Machinery TAP in Adaptive Immunity and Virus Escape Mechanisms

The transporter associated with antigen processing (TAP) is a crucial element of the adaptive immune system, which translocates proteasomal degradation products into the endoplasmic reticulum, for transfer of these peptides on major histocompatibility complex (MHC) I molecules within a macromolecular peptide-loading complex. After loading and intracellular transport to the cell surface, these peptide/MHC complexes are monitored by cytotoxic T-lymphocytes. This review summarizes the structural organization and function of the ABC transporter TAP. Furthermore, we discuss human diseases and viral evasion strategies associated with TAP function.