Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing D amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex͞T cell receptor complex. Cytotoxic T lymphocytes distinguish between self and non-self by monitoring peptides presented in association with major histocompatibility complex (MHC) class I molecules on the cell surface. These peptides are believed to be generated mainly from endogenous proteins in the proteasomal pathway, and they have to cross the membrane of the endoplasmic reticulum (ER) for association with assembling class I molecules. The essential role of the heterodimeric TAP (transporter associated with antigen processing) complex in peptide translocation across the ER membrane became evident from studies with mutant cell lines that were deficient in MHC class I-dependent antigen presentation when, in transfection experiments with tap genes, cytotoxic T cell recognition could be restored (1-3). ATP-dependent peptide translocation into the ER lumen by TAP was demonstrated directly, using streptolysin O-permeabilized cells (4, 5) or microsomal membranes (6, 7). In these experiments, transported peptides were retained in the ER by trapping on MHC class I molecules or through N-linked glycosylation of peptides carrying a consensus recognition site (NXS͞T). From these results more detailed questions about the immunological relevance of TAP arose: (i) does TAP put a restriction on the pool of antigenic peptides available for presentation to cytotoxic T cells, and (ii), if so, what is the peptide selectivity of TAP?Several assays have been developed to study peptide specificity of TAP. Most results came from assays relying on trapping of transported peptides in the ER via glycosylation. Comparing the amount of glycos...
Hepatitis C virus (HCV) is a major cause of parenterally Antibodies directed to hypervariable region 1 (HVR1) transmitted acute and chronic hepatitis. 1 Upon infection, paof hepatitis C virus (HCV) have recently been shown to tients develop a chronic persistent infection in more than neutralize the corresponding HCV isolate in vitro. We 50% of these cases. 2 Antibody responses 3 and T-cell-medianalyzed the appearance of antibodies directed to HVR1 ated immune responses to various regions of the HCV polyduring the course of infection in a large group of paprotein 4,5 have been studied, but so far a prognostic marker tients who have been infected by the same isolate of a associated with acute self-limited or persistent infections has HCV contaminated anti-D immunoglobulin (HCV-AD78).not been found. An enzyme-linked immunosorbent assay (ELISA) was esVaccination studies of chimpanzees have shown the importablished using a synthetic peptide to detect antibodies tance of the immune response against the putative HCV enagainst the main HVR1 variant of HCV-AD78. 207 sera velope proteins E1/E2. 6 However, protection of animals deobtained at different time points post infection (p.i.) of pends on the HCV isolate used for challenge of immunized 51 patients having either acute self-limiting (n Å 28) or animals. In addition, reinfection occurred in chimpanzees 7 chronic infection (n Å 23) were studied. Antibodies diand was suggested for patients. [8][9][10] Isolate-specific, neutralizrected to HVR1 were found at least at one time point ing antibodies directed against E1 and E2 may be involved during the infection course in 15 of 28 patients (53%) in determination of the course of infection. Antibodies present having acute self-limiting infections and in 17 of 23 pain chronically infected patients after several years postinfectients (74%) with chronic disease. The time of appeartion (p.i.) fail to neutralize the viral variant present in the ance of anti-HVR1 was significantly different between infectious source 11 most probably because of an immune esthese two patient groups (P õ .025) although appearance cape of newly arising viral variants of hypervariable region and titers of other HCV-specific antibodies were found 1 (HVR1). 12 Sequences of HVR1 found in earlier time points to be similar at early time points p.i. In acute self-limof infection are rapidly mutated during chronic infection and iting infections 9 of 21 sera (43%) of respective patients followed by respective, HVR1-specific antibody response. 13,14with sera available within the first 6 months p.i. were A monoclonal antibody directed against HVR1 was capable anti-HVR1 positive. The highest prevalence of antiof HCV in vitro neutralization. 15 Antibodies that neutralize HVR1 in this group of patients was within month 6 to HCV have also been observed in sera of HCV-infected pa-12 p.i. (64%). None of the sera available after 24 months tients. 11,16 Such neutralizing antibodies in patient sera are p.i. had such antibodies. In contrast, only 2 of 15 sera mainly direc...
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