Biomedical subjects

J J Neefjes

Publications and source records attributed to J J Neefjes.

At least 37 records · Page 2Linked to original sources

PubMed1994

Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments.

The HLA-DM genes encode an unconventional HLA (human leukocyte antigen) class II molecule that is required for appropriate binding of peptide to classical HLA class II products. In the absence of DM, other class II molecules are unstable upon electrophoresis in sodium dodecyl sulfate and are largely associated with a nested set of peptides derived from the invariant chain called CLIP, for class II-associated invariant chain peptides. DMA and DMB associated and accumulated in multilaminar, intracellular compartments with classical class II molecules, but were found infrequently, if at all, at the cell surface. Thus, DM may facilitate peptide binding to class II molecules within these intracellular compartments.

Animals↗

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

Targeting major histocompatibility complex class II molecules to the cell surface by invariant chain allows antigen presentation upon recycling.

We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class II molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (delta 15Ii) or 23 (delta 23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas delta 15Ii did not affect antigen presentation by class II molecules, delta 23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of delta 23Ii can be internalized, followed by degradation of delta 23Ii and return of free class II alpha beta heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.

Antigen Presentation↗

PubMed1994

Peptide selection by MHC-encoded TAP transporters.

With the discovery of MHC-encoded peptide transporters (TAP) came the identification of a new class of molecules within the immune system. TAP belongs to a large family of ATP-binding, multimembrane-spanning transporters that are expressed in a diversity of cells, from prokaryotic to mammalian, and show specificity for a variety of different substrates. TAP represents the solution to a major topological problem in immunology, namely the translocation of peptides, generated by cytosolic degradation of antigens, into the lumen of the endoplasmic reticulum where they associate with newly synthesized MHC class I molecules. A novel assay allows us to determine the requirements for the TAP-mediated peptide transport. First results indicate that TAP preselects peptides according to sequence and length in a way that is compatible with the characteristics of peptides isolated from class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Folding and assembly of major histocompatibility complex class I heterodimers in the endoplasmic reticulum of intact cells precedes the binding of peptide.

Major histocompatibility complex (MHC) class I molecules are heterotrimers consisting of a polymorphic H chain, beta 2-microglobulin (beta 2m) and peptide. Peptides are thought to associate early during biosynthesis but the order of assembly of class I molecules from their component subunits in intact cells is not settled. We have studied the assembly of MHC class I molecules in intact cells with or without peptide transporters. MHC class I H chain/beta 2m heterodimers can be efficiently recovered only 4 min after translation and are preceded by a folding intermediate. Approximately 2 min after their formation, the class I heterodimers are loaded with peptides resulting in stable class I heterotrimers. In these in vivo studies, no evidence was obtained that peptide binding to the H chain preceded the association with beta 2m. In contrast, nonassembled class I H chains could be recovered immediately after translation, but this pool did not participate in the formation of class I molecules.

Biological Transport↗

PubMed1993

Selective and ATP-dependent translocation of peptides by the MHC-encoded transporter.

Major histocompatibility complex (MHC) class I molecules present peptides derived from nuclear and cytosolic proteins to CD8+ T cells. These peptides are translocated into the lumen of the endoplasmic reticulum (ER) to associate with class I molecules. Two MHC-encoded putative transporter proteins, TAP1 and TAP2, are required for efficient assembly of class I molecules and presentation of endogenous peptides. Expression of TAP1 and TAP2 in a mutant cell line resulted in the delivery of an 11-amino acid oligomer model peptide to the ER. Peptide translocation depended on the sequence of the peptide, was adenosine triphosphate (ATP)-dependent, required ATP hydrolysis, and was inhibited in a concentration-dependent manner.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Phosphorylation of surface E-selectin and the effect of soluble ligand (sialyl Lewisx) on the half-life of E-selectin.

E-selectin (ELAM-1) is an adhesion molecule for leukocytes that is transiently expressed on endothelial cells. Following cell surface expression of E-selectin on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor, the induced E-selectin molecules are rapidly degraded. The kinetics of turnover of surface disposed E-selectin were investigated. The rapid disappearance of surface E-selectin is temperature dependent and sensitive to the lysosomotropic agent chloroquine. The half-life of E-selectin is not affected by inclusion of soluble sialyl Lewis x (sLex) ligands in the medium. Surface E-selectin is phosphorylated on one or more serine residues, but this modification is not obviously related to internalization.

Cell Adhesion Molecules↗

PubMed1993

The effect of anchor residue modifications on the stability of major histocompatibility complex class I-peptide interactions.

Anchor residues in peptides determine the specificity of binding to major histocompatibility complex class I molecules through interactions of their side chains with pockets in the peptide-binding groove. We have compared the kinetics of association of a Sendai virus nucleoprotein-derived peptide (FAPGNYPAL, termed SV9) with H-2Kb class I molecules, and the same peptide iodinated on the anchor residue tyrosine (125I-SV9). Even though the association rates were too rapid for direct measurements, competition studies indicated that they were similar for SV9 and 125I-SV9. To measure the binding of non-radioactive SV9 directly, SV9 was tritiated (3H-SV9). 3H-SV9 remained stably associated with H-2Kb molecules, whereas 125I-SV9 dissociated in a temperature-dependent fashion. Thus, modifications on anchor residues do not necessarily have to affect the specificity and association kinetics of peptide binding to class I molecules but can affect the stability of the resulting class I-peptide interaction. The dissociation of peptides with modified and, more generally, suboptimal anchor residue side chains may explain the presence of empty class I molecules and free class I heavy chains at the cell surface.

Amino Acid Sequence↗

PubMed1993

The assembly of H2-Kb class I molecules translated in vitro requires oxidized glutathione and peptide.

Association of the mouse major histocompatibility complex (MHC) class I heavy chain H2-Kb with mouse beta 2-microglobulin (beta 2m) was studied in an in vitro translation system. Formation of stable class I complexes was found to be dependent on the presence of presentable peptides and oxidized glutathione, which promotes the formation of disulfide bridges. Translocation of peptides into microsomes was demonstrated by showing that a radioiodinated peptide containing an N-glycosylation acceptor site became glycosylated. Class I complex formation was observed only when heavy chains and beta 2m were translated simultaneously, and thus occurs in the microsomes and not after their solubilization. However, peptide binding takes place only after solubilization of the microsomes. The class I complexes translated in vitro show the same specificity and length preference for peptides as their counterparts in RMA-S cells. Assembly of in vitro translated class I complexes was found to occur also in the absence of peptides, resulting in the formation of unstable molecules that are stabilized by incubation with peptides.

Amino Acid Sequence↗

PubMed1993

Cell biology of antigen presentation.

MHC class I molecules present degradation products derived from intracellular proteins, whereas MHC class II molecules generally present peptides derived from extracellular or surface proteins. Recent insights into the cell biology of MHC class I and II molecules explain this difference.

Animals↗

PubMed1992

The fate of the three subunits of major histocompatibility complex class I molecules.

At the surface of the murine T lymphoma cell line RMA-S, the expression of "empty" class I molecules can be dramatically enhanced by culture at 26 degrees C. These class I molecules are unstable following transfer to 37 degrees C unless they are loaded with exogenously added peptides. Class I heterodimers that have failed to bind peptide ("empty" class I molecules) dissociate and the class I heavy chains are degraded. Internalization, if it precedes breakdown, would be the rate-limiting step. Radioiodinated peptides (VSV NP 8-mer or Sendai NP 9-mer) dissociate from the class I molecules in the absence of exogenous peptide or beta 2 microglobulin and appear in the medium. Release of the iodinated peptides does not result in a reduction in the quantity of stable assembled class I molecules. This paradox may be explained by a more rapid off-rate for radioiodinated peptides, when compared with their unlabeled counterparts, which constitute about 99% of the total in our radiolabeled preparations. In the medium the peptide is rapidly modified by serum- and cell-derived proteases. The short half-life of empty class I molecules and of free ligand would effectively preclude sensitization of innocent bystanders for lysis by cytotoxic T lymphocytes.

Amino Acid Sequence↗

PubMed1992

HLA class I and II molecules present influenza virus antigens with different kinetics.

Human leukocyte antigen (HLA) class I and class II molecules differ with respect to their intracellular pathways and the compartments where they associate with processed antigen. To study possible consequences of these differences for the kinetics of antigen presentation by HLA class I and class II molecules, we analyzed changes in the concentrations of free intracellular calcium ions in influenza virus-specific T cell clones after recognition of specific antigen/HLA complexes. HLA class II-restricted viral antigen presentation by Epstein-Barr virus-transformed B lymphoblastoid cell lines (B-LCL) to CD4+ T cell clones started within 1 h and showed little variability, irrespective of antigen specificity or restriction element tested. In contrast, kinetics of viral antigen presentation by HLA class I molecules to CD8+ T cell clones were slower and differed for three antigen/HLA class I complexes tested. While B-LCL presented antigen by HLA-A2 and by HLA-B37 after at least 2 h, they only started to present antigen in the context of HLA-B7 after more than 4 h. This difference in kinetics did not correlate with differences in bulk transport rates of HLA-A2, HLA-B37, and HLA-B7, but seemed greatly influenced by differential rates of peptide generation. Brefeldin A treatment of B-LCL showed for both HLA class I and class II that de novo synthesized HLA molecules were involved in antigen presentation. Thus, differences between intracellular pathways of HLA class I and class II molecules may result in different kinetics of antigen presentation.

Antigens, Viral↗

PubMed1992

Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistance alpha beta heterodimers in endosomes.

The biosynthesis of MHC Class II molecules starts with the assembly of the alpha and beta subunits and the invariant chain. Intracellular transport of Class II molecules was followed in pulse-chase experiments of a human Epstein-Barr virus-transformed B lymphoblastoid cell line. Entry of Class II molecules into the endocytotic pathway and their cell surface appearance were monitored using neuraminidase as a fluid endocytotic marker and as a surface probe, respectively. In the course of intracellular transport, the Class II associated invariant chain is removed by proteases located in the endosomal pathway. Here, we show that leupeptin inhibits not only invariant chain breakdown, but also surface deposition of newly synthesized Class II molecules. Class II molecules display remarkable resistance to SDS at ambient temperature when occupied by peptide. We exploit this property to show that peptide binding precedes surface expression, and takes place in the course of intracellular transport through an endosomal compartment. Leupeptin blocks the conversion of Class II molecules to an SDS resistant complex.

Animals↗

PubMed1992

Intracellular transport of MHC class II molecules.

MHC class II molecules associate, during biosynthesis, with peptides derived from endocytosed antigen. Here, Jacques Neefjes and Hidde Ploegh describe the intracellular transport of MHC class II molecules and its relationship to the binding of peptides in endosomal compartments. They discuss alternative routes for the delivery of antigen to sites at which peptides associate with MHC class II molecules and raise the possibility of cell type-specific differences in the handling of MHC class II molecules, and hence in antigen presentation.

Animals↗

PubMed1992

Distribution and elimination of the glycosidase inhibitors 1-deoxymannojirimycin and N-methyl-1-deoxynojirimycin in the rat in vivo.

We studied the pharmacokinetics of two synthetic derivatives of 1-deoxynojirimycin in the rat after intravenous administration. The mannosidase IA/B inhibitor 1-deoxymannojirimycin and the glucosidase inhibitor N-methyl-1-deoxynojirimycin exhibited minimal plasma protein binding and showed a rapid biphasic plasma disappearance, with an initial t1/2 of 3.0 and 4.5 min, respectively, and a terminal t1/2 of 51 and 32 min, respectively. For both compounds renal excretion is the major route of elimination. After 120 min, 52% of the dose of 1-deoxymannojirimycin and 80% of the dose of N-methyl-1-deoxymannojirimycin was recovered unchanged from the urine, whereas only 4.9 and 0.2%, respectively, of the dose was excreted in bile. Urinary clearance of 1-deoxymannojirimycin was similar to the glomerular filtration rate. In contrast, urinary clearance of N-methyl-1-deoxynojirimycin was two to three times higher than the glomerular filtration rate, indicating active tubular secretion. Ligation of the renal vessels decreased the total-body clearance of 1-deoxymannojirimycin and N-methyl-1-deoxynojirimycin 18- and 24-fold, respectively. Neither alkalinization of the urine by infusion of bicarbonate solutions nor forced diuresis altered the renal excretion rate of these compounds, implying the absence of tubular reabsorption. At 120 min, the amounts of 1-deoxymannojirimycin in liver and kidney were 2.1 and 1.1% of the dose, respectively, while small intestine, stomach, and heart contained only 0.9, 0.6 and 0.1%. Less than 1% of the dose of N-methyl-1-deoxynojirimycin was found in the collected organs 2 hr after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Deoxynojirimycin↗

PubMed1992

E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro.

Endothelial cells respond to several cytokines by a rapid increase in expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1), followed by a gradual decline. The fate of these molecules, which was so far unknown, was studied. Specific sandwich ELISA for the detection of soluble (s)E-selectin and sICAM-1 were developed. In supernatant, centrifuged 3 hr at 100,000 g to remove microparticles, from human umbilical vein endothelial cells (HUVEC) activated with tumour necrosis factor (TNF), interleukin-1 (IL-1) or lipopolysaccharide (LPS), E-selectin and ICAM-1 molecules could be detected. Biochemical analysis revealed that sE-selectin migrated as a band of approximately 94,000 MW. The amount of soluble adhesion molecules released was directly correlated with cell surface expression. Maximal release of E-selectin was observed 6-12 hr after activation of HUVEC and decreased to below detection limit 24 hr after activation. After activation, release of ICAM-1 gradually increased with ICAM-1 cell surface expression, and reached a plateau after 24 hr, which was constant for 3 days. Since E-selectin and ICAM-1 are highly expressed at inflammatory sites, the resulting high concentrations of released E-selectin and ICAM-1 may affect interactions of leucocytes with endothelial cells. The physiological role, however, of the release of E-selectin and ICAM-1 remains to be elucidated.

Antibodies, Monoclonal↗

PubMed1991

Peptide selection by MHC class I molecules.

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.

Amino Acid Sequence↗