Modulation of the antigenic peptide transporter TAP by recombinant antibodies binding to the last five residues of TAP1

The transporter associated with antigen processing (TAP) plays a pivotal role in the major histocompatibility complex (MHC) class I mediated immune response against infected or malignantly transformed cells. It belongs to the ATP-binding cassette (ABC) superfamily and consists of TAP1 (ABCB2) and TAP2 (ABCB3), each of which possesses a transmembrane and a nucleotide-binding domain (NBD). Here we describe the generation of recombinant Fv and Fab antibody fragments to human TAP from a hybridoma cell line expressing the TAP1-specific monoclonal antibody mAb148.3. The epitope of the antibody was mapped to the very last five C-terminal amino acid residues of TAP1 on solid-supported peptide arrays. The recombinant antibody fragments were heterologously expressed in Escherichia coli and purified to homogeneity from periplasmic extracts by affinity chromatography. The monoclonal and recombinant antibodies bind with nanomolar affinity to the last five C-terminal amino acid residues of TAP1 as demonstrated by ELISA and surface plasmon resonance. Strikingly, the recombinant antibody fragments confer thermal stability to the heterodimeric TAP complex. At the same time TAP is arrested in a peptide transport incompetent conformation, although ATP and peptide binding to TAP are not affected. Based on our results we suggest that the C terminus of TAP1 modulates TAP function presumably as part of the dimer interface of the NBDs.     

Biogenesis of functional antigenic peptide transporter TAP requires assembly of pre-existing TAP1 with newly synthesized TAP2

The transporter associated with antigen processing (TAP) is essential for the delivery of antigenic peptides from the cytosol into the endoplasmic reticulum (ER), where they are loaded onto major histocompatibility complex class I molecules. TAP is a heterodimeric transmembrane protein that comprises the homologous subunits TAP1 and TAP2. As for many other oligomeric protein complexes, which are synthesized in the ER, the process of subunit assembly is essential for TAP to attain a native functional state. Here, we have analyzed the individual requirements of TAP1 and TAP2 for the formation of a functional TAP complex. Unlike TAP1, TAP2 is very unstable when expressed in isolation. We show that heterodimerization of TAP subunits is required for maintaining a stable level of TAP2. By using an in vitro expression system we demonstrate that the biogenesis of functional TAP depends on the assembly of preexisting TAP1 with newly synthesized TAP2, but not vice versa. The pore forming core transmembrane domain (core TMD) of in vitro expressed TAP2 is necessary and sufficient to allow functional complex formation with pre-existing TAP1. We propose that the observed assembly mechanism of TAP protects newly synthesized TAP2 from rapid degradation and controls the number of transport active transporter molecules. Our findings open up new possibilities to investigate functional and structural properties of TAP and provide a powerful model system to address the biosynthetic assembly of oligomeric transmembrane proteins in the ER.     

EB病毒蛋白BNLF2a阻止抗原转运蛋白TAP的构象变化

【背景】EB病毒是一个常见的病原,它能引起霍奇金淋巴瘤、伯基特淋巴瘤以及胃癌、鼻咽癌。该病毒编码的膜蛋白BNLF2a抑制抗原转运蛋白TAP (Transporter associated with antigen processing)从而逃逸T细胞的清除。TAP属于ABC(ATP-bindingcassette)转运蛋白超家族,是由TAP1和TAP2两个亚基构成的。TAP通过ATP提供能量,跨膜转运抗原多肽,这一过程伴随着构象变化。【目的】旨在揭示BNLF2a是否影响TAP的构象变化。【方法】TAP蛋白核酸结合结构域的二聚体界面的D-loop进行点突变,引入半胱氨酸。在表达和不表达BNLF2a情况下,采用氧化性的二价铜离子交联半胱氨酸,并通过Westernblot对比TAP的半胱氨酸形成二硫键的比例。【结果】BNLF2a表达使TAP被交联的比例增高。【结论】BNLF2a可能将TAP稳定在核苷酸结合结构域二聚化的构象,从而同时抑制ATP和抗原多肽结合到TAP上来。     

The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding

The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.     

Thermodynamics of peptide binding to the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP plays an essential role in antigen processing and immune response to infected or malignant cells. TAP translocates proteasomal degradation products from the cytosol into the endoplasmic reticulum, where MHC class I molecules are loaded with these peptides. Kinetically stable peptide-MHC complexes are transported to the cell surface for inspection by cytotoxic T lymphocytes. The transport cycle of TAP is initiated by peptide binding, which is responsible for peptide selection and for stimulation of ATP-hydrolysis and subsequent translocation. Here we have analysed the driving forces for the formation of the peptide-TAP complex by kinetic and thermodynamic methods. First, the apparent peptide association and dissociation rates were determined at various temperatures. Strikingly, very high activation energies for apparent association (E(a)(ass)=106 kJmol(-1)) and dissociation (E(a)(diss)=80 kJmol(-1)) of the peptide-TAP complex were found. Next, the temperature-dependence of the peptide affinity constants was investigated by equilibrium-binding assays. Along with calculations of free enthalpy deltaG, enthalpy deltaH and entropy deltaS, a large positive change in heat capacity was resolved (deltaC degrees =23 kJmol(-1)K(-1)), indicating a fundamental structural reorganization of the TAP complex upon peptide binding. The inspection of the conformational entropy reveals that approximately one-fourth of all TAP residues is rearranged. These thermodynamic studies indicate that at physiological temperature, peptide binding is endothermic and driven by entropy.     

Impaired transporter associated with antigen processing (TAP) function attributable to a single amino acid alteration in the peptide TAP subunit TAP1

The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1(-/-) cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu(263) was comparable to that of TAP1(-/-) cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe(265) of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu(263) of the TAP1 protein, but not the Phe(265), is critical for proper TAP function.     

Functionally important interactions between the nucleotide-binding domains of an antigenic peptide transporter

The transporter associated with antigen processing (TAP), an ABC transporter, pumps cytosolic peptides into the endoplasmic reticulum, where the peptides are loaded onto class I MHC molecules for presentation to the immune system. Transport is fueled by the binding of ATP to two cytosolic nucleotide-binding domains (NBDs) and ATP hydrolysis. We demonstrate biochemically that there are two electrostatic interactions across the interface between the two TAP NBDs and that these interactions are important for peptide transport. Notably, disrupting these interactions by mutagenesis does not greatly alter the ATP hydrolysis rate in an isolated NBD model system, suggesting that the interactions function at alternative stages in the transport cycle. The data support the general model for ABC transporters in which the NBDs form a tight, closed conformation during transport. Our results are discussed in relation to other ABC transporters that do or do not conserve potential interacting residues of opposite charges at the homologous positions.     

Hypothyroidism induces expression of the peptide transporter PEPT2

The kidney is a target organ for thyroid hormone action and a variety of renal transport processes are altered in response to impaired thyroid functions. To investigate the effect of thyroid hormone on the expression of the renal proximal tubular high-affinity-type H(+)-peptide cotransporter (PEPT2) in rats, hypothyroidism was induced in animals by administration of methimazole (0.05%) via drinking water. After 7 weeks of treatment, hypothyroidism was confirmed by determining serum free T(3) and free T(4) concentrations. Northern blotting was used to examine the expression of PEPT2 mRNA in kidney tissues from hypothyroid rats compared to control rats. Hypothyroidism resulted in an increased level of total renal PEPT2 mRNA (121.1+/-3.3% vs. control 100+/-2.8%; p=0.008). The mRNA results were confirmed by immuno-blotting, which demonstrated significantly increased protein levels (162% vs. control 100%; p<0.01). Immunohistochemistry also revealed increased PEPT2 protein levels in the proximal tubules of treated compared to non-treated rats. In summary, PEPT2 is the first proximal tubule transporter protein that shows increased expression in states of hypothyreosis. As PEPT2 reabsorbs filtered di- and tripeptides and peptide-like drugs, the present findings may have important implications in nutritional amino acid homeostasis and for drug dynamics in states of altered thyroid function.     

Modulation of transporter associated with antigen processing (TAP)-mediated peptide import into the endoplasmic reticulum by flavivirus infection

In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207-214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.     

In vivo augmentation of tumor-specific CTL responses by class I/peptide antigen complexes on microspheres (large multivalent immunogen)

Tumor membrane Ag immobilized on cell size microspheres (large multivalent immunogen (LMI)) was previously shown to augment tumor-specific CTL activity and reduce tumor growth, and a clinical trial examining this approach is in progress. In the current study, LMI treatment has been examined using adoptive transfer of TCR-transgenic CD8 T cells to visualize Ag-specific cells during the response. OT-I T cells specific for H-2K(b)/OVA(257-264) were transferred into mice that were then challenged with LMI made by immobilizing H-2K(b)/OVA(257-264) on microspheres (K(b)/OVA(257-264)-LMI) alone, or along with i.p. challenge with OVA-expressing E.G7 tumor. K(b)/OVA(257-264)-LMI caused significant reduction of tumor growth when administered to E.G7-bearing mice. When administered alone, the K(b)/OVA(257-264)-LMI caused only weak clonal expansion of OT-I cells in the spleen and lymph nodes, although most of the OT-I cells up-regulated expression of CD44 and VLA-4. In contrast, K(b)/OVA(257-264)-LMI administration to E.G7-bearing mice stimulated no detectable expansion of OT-I cells in the spleen and lymph nodes but caused a rapid increase in the number of OT-I cells in the peritoneal cavity, the site of the growing tumor. These results demonstrate the potential for using class I/tumor peptide complexes for immunotherapy. In addition, they suggest a model for the mechanism of CTL augmentation in which recognition of the LMI Ag results in altered trafficking of the tumor-specific CD8 T cells so that they reach the site of a growing tumor more rapidly and in greater numbers, where they may further expand and acquire effector function.     

Simultaneous exploitation of different peptide permeases by combinations of synthetic peptide smugglins can lead to enhanced antibacterial activity

Necrotizing Escherichia coli (NTEC) strains grown in the presence of mitomycin C released cell associated necrotizing factors CNF1 and CNF2 to culture medium. Using culture filtrates from 96 mitomycin C treated E. coli strains, we have found that a modified HeLa cell assay was a more sensitive and specific method for the detection of CNF1 and CNF2 than the Vero cell assay and the rabbit skin test.     

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.     

Preferential recognition of zwitterionic dipeptides as transportable substrates by the high-affinity peptide transporter PEPT2.

We investigated the interaction of rat PEPT2, a high-affinity peptide transporter, with neutral, anionic, and cationic dipeptides using electrophysiological approaches as well as tracer uptake methods. D-Phe-L-Gln (neutral), D-Phe-L-Glu (anionic), and D-Phe-L-Lys (cationic) were used as representative, non-hydrolyzable, dipeptides. All three dipeptides induced H+-dependent inward currents in Xenopus laevis oocytes heterologously expressing rat PEPT2. The H+:peptide stoichiometry was 1:1 in each case. A simultaneous measurement of radiolabeled dipeptide influx and charge transfer in the same oocyte indicated a transfer of one net positive charge into the oocyte per transfer of one peptide molecule irrespective of the charged nature of the peptide. We conclude that the zwitterionic peptides are preferentially recognized by PEPT2 as transportable substrates and that the proton/peptide stoichiometry is 1 for the transport process.     

Cutting edge: cross-presentation as a mechanism for efficient recruitment of tumor-specific CTL to the brain

The number and localization of effector cells to the tumor site are crucial elements for immune rejection of solid tumors. However, for cerebral malignancies, antitumor responses need to be finely tuned to avoid neuropathologic consequences. In this study, we determine factors that regulate CTL localization and tumoricidal function after intracerebral implantation of tumors expressing model Ag. H-2(bxd) mice implanted with a CW3(+) murine glioma lacking H-2K(d) molecules necessary to present the CW3(170-179) epitope demonstrate cross-priming of H-2K(d)-restricted CTL, and moreover, Ag-dependent accumulation of functional H-2K(d)/CW3(170-179)-specific CTL within the tumor bed. This implicates a role for cross-presentation not only in priming, but also in retention of fully differentiated CTL in the tumor stroma at the effector stage of the response. Modulating cross-presentation of Ag may be the key in regulating specific immune responses in the brain: either by augmenting protective responses or by down-modulating destructive autoimmune reactions.     

T cell receptor junctional regions and the MHC molecule affect the recognition of antigenic peptides by T cell clones.

Nine independent pigeon cytochrome c-specific T cell clones were analyzed by using a panel of antigenic peptide analogs presented in association with three allelic IE-encoded MHC glycoproteins. Eight of the T cell clones expressed a TCR composed of a unique alpha- and beta-chain amino acid sequence, and concordantly, each of these T cell clones exhibited a unique Ag specificity. This was true for several clones which differed only in TCR V-J junctional regions. Interestingly, for a given clone, the response to some of the peptide analogs depended to a large extent on the allelic form of the presenting MHC molecule. A simple interpretation of these data would suggest that certain positions of the peptide Ag are most important for Ag-MHC molecule interactions, and that these specific interactions can influence the antigenic epitope recognized by the TCR. We suggest that an antigenic peptide binds to an MHC glycoprotein in a distinct way, but may retain a measure of flexibility.     

TAP expression provides a general method for improving the recognition of malignant cells in vivo

A major class of tumors lack expression of the transporters associated with antigen processing (TAP). These proteins are essential for delivery of antigenic peptides into the lumen of the endoplasmic reticulum (ER) and subsequent assembly with nascent major histocompatibility complex (MHC) class I, which results in cell surface presentation of the trimeric complex to cytolytic T lymphocytes. Cytolytic T lymphocytes are major effector cells in immunosurveillance against tumors. Here we have tested the hypothesis that TAP downregulation in tumors allows immunosubversion of this effector mechanism, by establishing a model system to examine the role of TAP in vivo in restoring antigen presentation, immune recognition, and effects on malignancy of the TAP-deficient small-cell lung carcinoma, CMT.64. To test the potential of providing exogenous TAP in cancer therapies, we constructed a vaccinia virus (VV) containing the TAP1 gene and examined whether VV-TAP1 could reduce tumors in mice. The results demonstrate that TAP should be considered for inclusion in cancer therapies, as it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic complement of the tumor or the MHC haplotypes of the host.     

Soil amendment application frequency contributes to phytoextraction of lead by sunflower at different nutrient levels

Phytoextraction has been proposed as an alternative remediation technology for soils polluted with heavy metals, but is generally perceived to be too slow. Enhancing accumulation of trace pollutants in harvestable plant tissues is a prerequisite for such technology to be practical. The main aims of this paper were to investigate whether a combination of nutrients and ethylenediaminetetraacetic acid (EDTA) enhanced Pb uptake of sunflower (Helianthus annuus) plants, and if timing of EDTA application altered Pb uptake and environmental persistence. Plants were grown in greenhouse pot experiments. Pb distributions and uptake of the whole plant were studied using chemical and flame atomic absorption spectrometry analyses. Pb mobilization by EDTA appeared to be dose dependent, with more mobilization for the high than the low dose. There were distinct differences in mobilization patterns of various nutrient amendments. EDTA mobilized Pb more in the medium than the highest and lowest nutrient levels. Heterogeneous soil humus components exerted mobilizing and stabilizing effects, so the medium nutrition was most effective for phytoextraction. At low nutrient levels, Pb concentration in the shoot with one low EDTA application was less than two applications to the same total EDTA dosage. So in the poor soil, two applications of EDTA was more effective than once. The half-life of two low EDTA treatment applications was longer than for one application, to the same total dosage. In general, sunflower was suited to phytoremediation of moderately Pb-contaminated soil by phytoextraction.     

The varicellovirus-encoded TAP inhibitor UL49.5 regulates the presentation of CTL epitopes by Qa-1b1

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1(b) revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.