Biomedical subjects

J Roelse

Publications and source records attributed to J Roelse.

8 recordsLinked to original sources

PubMed1995

Major differences in transporter associated with antigen presentation (TAP)-dependent translocation of MHC class I-presentable peptides and the effect of flanking sequences.

The MHC-encoded transporter associated with Ag presentation (TAP) translocates peptides from the cytosol to the ER lumen, where association with MHC class I molecules occurs. The MHC class I/peptide complex is subsequently transported to the cell surface for presentation to CD8+T cells. We studied TAP-dependent translocation of defined MHC class I presentable murine peptides by competition for translocation of a radiolabeled model peptide, to address whether efficient peptide presentation by MHC class I molecules is preceded by equal efficient peptide translocation by TAP. Surprisingly, we observed that four immunodominant viral peptides of 16 peptides tested were very inefficiently transported by TAP. Inefficient translocation could be overcome by substitution of a proline residue present at position 3 in the peptides. Furthermore, addition of natural flanking amino acids directly surrounding a poorly transported peptide could considerably improve translocation by TAP. Our data suggest that some peptides are efficiently transported by TAP in their optimal size for MHC class I binding, whereas other peptides are transported as larger peptide fragments that need further trimming in the ER for MHC class I binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Analysis of the fine specificity of rat, mouse and human TAP peptide transporters.

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling.

Cytosolic peptides are translocated to the endoplasmic reticulum (ER) lumen by the transporters associated with antigen processing (TAP), where major histocompatibility complex (MHC) class I molecules associate with peptides of about 8-10 amino acids. TAP translocates peptides of 9-13 amino acids with the highest relative affinity but also longer and shorter peptides. The fate of the peptides that fail to associate with class I molecules because of incorrect sequence or length, is unknown. Here we show that the bulk of the translocated peptides are rapidly released from the ER by a mechanism that requires adenosine triphosphate (ATP) and that could not be inhibited by GTP gamma S. TAP does not appear to be involved in this process. Whereas free peptides are slowly trimmed in the ER lumen, they are rapidly degraded in the cytosol. A fraction of the peptides released from the ER escapes complete degradation in the cytosol and recycles back to the ER in a TAP-dependent fashion. These results suggest that peptides that are too long for binding to class I molecules in the ER can be trimmed further in the ER lumen or, alternatively, can be transported back to the cytosol where a fraction of the peptides is trimmed to a size suitable for association to MHC class I molecules and recycles back to the ER.

ATP-Binding Cassette Transporters↗

PubMed1994

The stability and steady-state kinetics of vanadium chloroperoxidase from the fungus Curvularia inaequalis.

In this article we report on the steady-state kinetics of the chlorination and the stability of the vanadium chloroperoxidase from the fungus Curvularia inaequalis. The data show that the kinetics of this enzyme resemble that of the vanadium bromoperoxidase from the seaweed Ascophyllum nodosum. At low pH, chloride inhibited the enzyme, but the inhibition was of a dual nature. At pH 4.1 a mixed type of inhibition by chloride with respect to hydrogen peroxide was observed whereas at pH 3.1 the nature of the inhibition became competitive. The log Km for hydrogen peroxide decreased linearly with pH with a slope of -1 in the pH range 3-5. A reaction mechanism is presented to explain the observed data. We also showed that this class of enzymes is inhibited by nitrate. At pH 5.5, nitrate inhibits the chlorination reaction competitively with respect to chloride (Ki = 2 mM) and uncompetitively with respect to hydrogen peroxide. Furthermore, we showed that the enzyme produces HOCl as a reaction product. The enzyme exhibited a high thermostability (tm of 90 degrees C) and displayed high stability in organic solvents (solutions of 40% methanol, ethanol or 2-propanol) and moderate stability in the presence of the chaotropic agent guanidine/HCl (G1/2, the concentration of guanidine/HCl at which the enzyme activity was half the original activity was 3.7 M).

Chloride Peroxidase↗

PubMed1994

Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V.

Abnormal coagulation factor V may underlie the thrombotic events associated with resistance to activated protein C (APC). We analysed 27 consecutive patients with documented idiopathic (recurrent) thromboembolism for the occurrence of point mutations within the APC sensitive regions of blood coagulation factor V. In 10 patients we observed a single basepair mutation resulting in a substitution of Arg506 to Gln. This mutation was significantly linked to in-vitro resistance to APC in these subjects. This mutation at Arg506 of factor V may form the molecular basis for the thrombotic events associated with APC resistance.

Adult↗

PubMed1994

Peptide size selection by the major histocompatibility complex-encoded peptide transporter.

The major histocompatibility complex (MHC)-encoded heterodimeric TAP1/TAP2 transporter (TAP) translocates cytosolic peptides into the lumen of the endoplasmic reticulum (ER), where peptides of 8 to 11 amino acids long associate with MHC class I molecules. We have studied the selectivity of peptide translocation by TAP in streptolysin O-permeabilized cells using glycosylatable, radioiodinated model peptides to detect import into the ER lumen. TAP-dependent translocation of a radiolabeled nonamer peptide was most efficiently inhibited by unlabeled 9- to 11-mer peptides. Peptides between 7 and 40 amino acids long all could inhibit transport, the longer peptides being least effective. Also, peptides shorter than eight amino acids were inefficiently translocated. The use of directly labeled length variants in translocation assays and TLC analysis of the transported material revealed two pathways for translocation: short peptides (7 to 13 amino acids long) were translocated without prior modification. In contrast, transport of longer peptides was not effective. Instead such peptides were clipped by cytosolic peptidases before efficient transport. Our data suggest that TAP preferentially translocates peptides of appropriate length for class I binding. Furthermore, TAP-translocated peptides were rapidly released from the ER unless they were trapped there by being glycosylated or by binding to MHC class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Selectivity of MHC-encoded peptide transporters from human, mouse and rat.

Major histocompatibility complex (MHC) class I molecules present peptides from degraded intracellular antigens to CD8+ T cells. These peptides are translocated in an ATP-dependent fashion into the lumen of the endoplasmic reticulum (ER) for binding to class I molecules by means of the MHC-encoded transporters associated with antigen processing, TAP1 and TAP2. These are members of a family of proteins containing an ATP-binding cassette and form heterodimers in the ER membrane. Defects in the genes encoding TAP1 or TAP2 account for impaired class I assembly and antigen presentation in several human and rodent cell lines. Whereas MHC class I molecules select peptides according to binding motifs, it is not clear to what extent the TAP1-TAP2 transporters have peptide sequence and length specificity. Previous studies of the rat MHC class I molecule RT1Aa, suggested a specific conveyance of peptides by rat TAP1-TAP2. Here we substitute the amino- and carboxy-terminal and the penultimate amino-acid residues of model peptides to show that these residues influence the efficiency of transport. Human TAP and rat TAPa translocated peptides with hydrophobic and basic C termini, whereas mouse TAP and rat TAPu preferred peptides with hydrophobic C termini. This pattern correlates with the predominant peptide binding profiles of mouse and human class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Peptide transporters and antigen processing.

Our knowledge about intracellular pathways involved in the presentation of antigens was considerably broadened with the recent discovery of peptide transporters encoded in the major histocompatibility complex. The transporter associated with antigen processing (TAP) belongs to an evolutionary conserved family of multimembrane-spanning translocators that bind ATP and show specificity for a variety of different substrates. TAP mediates the translocation of peptides, generated by cytosolic degradation of protein antigens, into the lumen of the endoplasmic reticulum where they bind to newly synthesized MHC class I molecules. A novel assay has been employed to elucidate the details of TAP-mediated peptide transport. The results indicate that TAP selects peptides of sequence and length according to the requirements of MHC class I molecules in different species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗