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O Bakke

Publications and source records attributed to O Bakke.

At least 37 records · Page 2Linked to original sources
PubMed1998

The leucine-based motif DDQxxLI is recognized both for internalization and basolateral sorting of invariant chain in MDCK cells.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains signals for transport to endocytic compartments where the class II molecules bind antigenic peptides for presentation to CD4+ T cells. Two leucine-based signals in the Ii cytoplasmic tail can be independently recognized for endosomal sorting of Ii, and we have recently shown that each signal is sufficient for basolateral sorting and internalization of Ii in polarized Madine Darby Canine Kidney (MDCK) II cells. The recognition motif for endosomal sorting is complex and consists of two critical leucine-like residues as well as surrounding amino acids. Here, we have analyzed the importance of residues surrounding the membrane-distal leucine-based signal in basolateral sorting and internalization of Ii in MDCK II cells. We find that the DDQxxLI motif is involved in both sorting events indicating the presence of similar signal recognition components both at the TGN and at the plasma membrane. The identical motif is required for endosomal localization and internalization of Ii also in simian COS cells and the human HeLa and M1 cells.

Amino Acid Sequence↗
PubMed1998

Selection of phage displayed peptides from a random 10-mer library recognising a peptide target.

BACKGROUND: Peptide display libraries are powerful tools in the search for detailed information about protein-protein interactions. Usual targets for isolation of phage displayed peptide ligands include antibodies, various receptors, other full size proteins or larger fragments thereof. Smaller protein fragments such as synthetic peptides have not been reported as targets for screening of peptide display libraries. OBJECTIVES: To investigate whether a protein target used for screening of a peptide display library could be scaled down to peptide size. As the peptide target we wanted to use a sequence derived from the cytosolic tail of MHC class II associated invariant chain containing a leucine class endosomal sorting signal, known to be recognised as an autonomous functional unit during targeting of class II complexes to antigen processing compartments. STUDY DESIGN: A screening procedure where a synthetic 15-mer invariant chain peptide was coupled to a methacrylate matrix of high binding capacity was developed, and three rounds of selection were performed from a random 10-mer fUSE5 display library. RESULTS: The peptide display library was successfully enriched for phage clones with affinity for the invariant chain peptide. Furthermore, the binding phage clones were able to distinguish between a functional and a mutated form of the target. These clones therefore displayed possible peptide mimetics of signal recognition sites in the cellular sorting machinery. CONCLUSION: The size of a protein target may be scaled down to peptide size and be recognised by a 10-mer peptide displayed on filamentous phage. This approach may particularly be useful when the peptide target contains a functional unit for recognition.

Amino Acid Sequence↗
PubMed1997

A new triple-stranded alpha-helical bundle in solution: the assembling of the cytosolic tail of MHC-associated invariant chain.

BACKGROUND: The invariant chain (li) is a transmembrane protein that associates with the major histocompatibility complex class II (MHC II) molecules in the endoplasmic reticulum. The cytosolic tail of li contains two leucine-based sorting motifs and is involved in sorting the MHC II molecules to the endosomal pathway where the peptide antigen is bound. This region of li also contributes to phenotypical changes in cells, such as the formation of large endocytic structures. RESULTS: We report here the three-dimensional structure of a 27 amino acid peptide corresponding to the cytosolic tail of li. The structure was determined by nuclear magnetic resonance (NMR) spectroscopy using a computational strategy. At high concentration, this structure reveals a new triple-stranded alpha-helical bundle in which the helices, two parallel and one antiparallel, are almost coplanar. Trimerization is mediated by electrostatic interactions intercalated by three hydrophobic layers. CONCLUSIONS: The new trimer fold, the first to be identified by NMR data alone, can be used to improve understanding of protein-protein interactions and to model multiple-helical transmembrane proteins and receptors. We suggest that interactions of the li cytosolic tails may form part of a mechanism that could cause the endosomal retention and enlarged endosomes induced by li.

Amino Acid Sequence↗
PubMed1997

MHC class II-associated invariant chain-induced enlarged endosomal structures: a morphological study.

The major histocompatibility complex class II-associated invariant chain is believed to direct newly synthesized class II to endocytic compartments. Invariant chain synthesized at high levels in transiently transfected cells induces formation of large vesicular structures. We have examined the effect of stable expression of invariant chain in human fibroblasts by light and electron microscopy. Invariant chain expression dramatically modified endocytic compartments and induced the formation of greatly enlarged structures. These modifications were not lethal. Ultrastructurally, at least three morphologically distinct enlarged compartments could be discerned in the cells. These three compartments may represent early and late endosomes and lysosomes. Internalization of anti-invariant chain antibodies shows that invariant chain may reach the large endosomes via rapid internalization from the plasma membrane. Internalized protein remained in the enlarged vesicles for 4-6 h, indicating an invariant chain-induced delay in the pathway to lysosomes. Although the large invariant chain-induced vesicles have not yet been seen in professional antigen-presenting cells, the invariant chain-induced effects may play a role in regulating the endocytic pathway, creating a special environment for MHC class II to bind antigen.

Adenine↗
PubMed1997

Exon 6 is essential for invariant chain trimerization and induction of large endosomal structures.

Invariant chain (Ii) is a transmembrane type II protein that forms a complex with the major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum (ER). The membrane proximal luminal region of Ii is responsible for the non-covalent association with MHC class II molecules. Chemical cross-linking in COS cells was used to study the effect of luminal and cytoplasmic deletions on trimerization of Ii. We demonstrate that trimerization of Ii is independent of the cytosolic tail of Ii, whereas residues 162-191 (the sequence encoded by exon 6) in the luminal part of Ii are essential for trimer formation. Immunofluorescence studies of the transfected luminal deletion constructs show that the amino acids encoded by exon 6 of Ii are also essential for the induction of large endosomal vesicles. The data suggest that Ii must be in a trimeric form to modify the endosomal pathway.

Amino Acid Sequence↗
PubMed1997

Sorting of MHC class II molecules and the associated invariant chain (Ii) in polarized MDCK cells.

Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the basolateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells.

Amino Acid Sequence↗
PubMed1995

Structure-activity relationship of the leucine-based sorting motifs in the cytosolic tail of the major histocompatibility complex-associated invariant chain.

The cytosolic tail of the major histocompatibility complex-associated invariant chain protein contains two Leu-based motifs that both mediate efficient sorting to the endocytic pathway. Nuclear magnetic resonance data on a peptide of 27 residues corresponding to the cytosolic tail of human invariant chain indicate that in water at pH 7.4 the membrane distal motif Leu7-Ile8 lies within a nascent helix, while the membrane proximal motif Met16-Leu17 is part of a turn. The presence of a small amount of methanol stabilizes an alpha helix from Gln4 to Leu17 with a kink on Pro15. Point mutations of the cytosolic tail of the protein suggest that amino-terminal residues located in spatial proximity to the Leu motifs contribute to efficient internalization and targeting to endosomes in transfected COS cells. Residues on the spatially opposite side of the Leu motifs were, on the other hand, mutated with no measurable effect on targeting. Structural and biological data thus suggest that the signals are not continuous but consist of "signal patches" formed by the three-dimensional structure of the cytosolic tail of invariant chain.

Amino Acid Sequence↗
PubMed1995

Antigen presentation mediated by recycling of surface HLA-DR molecules.

Class II histocompatibility molecules associate with peptides derived from antigens that are processed in endocytic compartments. Antigen presentation to class II-restricted T cells generally requires newly synthesized class II molecules, associated invariant chain, and HLA-DM. Exceptions to these rules have been reported, but without description of an underlying mechanism. Here we show that presentation of immunodominant epitopes in the haemagglutinin protein of influenza virus and in myelin basic protein correlates with recycling of surface HLA-DR molecules. Truncation of either one of the alpha or beta cytoplasmic tails virtually eliminated internalization of HLA-DR molecules and presentation of haemagglutinin from inactive virus particles. In contrast, the invariant chain-dependent presentation of matrix antigen from the same virus particles was unaffected by these truncations. Thus HLA-DR cytoplasmic tails are not required for the conventional presentation pathway, but jointly contribute a signal for an alternative pathway involving internalization of HLA-DR molecules.

Amino Acid Sequence↗
PubMed1995

Invariant chain induces a delayed transport from early to late endosomes.

Invariant chain associated with class II molecules is proteolytically processed in several distinct intermediates during its transport through the endocytic pathway. Using subcellular fractionation, early and late endosomal compartments were separated in human fibroblasts transfected with HLA-DR (4N5 cells) and supertransfected with invariant chain (4N5Ii cells) or invariant chain lacking most of the cytoplasmic tail (4N5 delta 20Ii cells). Early and late endosome membrane fractions were characterized by morphology and by analyzing the presence of the Rab5 and Rab7 GTPases as markers of early and late endosomes, respectively. The transfer of endocytosed horseradish peroxidase from early to late endosomes proceeded relatively rapid both in 4N5 and 4N5 delta 20Ii cells (t1/2 = 25 min), whereas this transfer was significantly delayed (t1/2 = 2 h) in 4N5Ii cells. Pulse-chase experiments showed that invariant chain and its degradation products were first observed in early endosomes and thereafter in late endosomes. Our results strongly suggest that invariant chain induces a retention mechanism in the endocytic pathway.

Antigens, Differentiation, B-Lymphocyte↗
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

The invariant chain inhibits presentation of endogenous antigens by a human fibroblast cell line.

The human fibroblast cell line, M1, expressing the products of transfected DRA and DRB1*0101 genes (M1-DR1) was unable to present intact influenza antigens to a series of DR1-restricted human T cell lines and clones, but was fully able to present synthetic peptides for T cell recognition. In contrast, M1-DR1 cells infected with live influenza virus were recognized by two polyclonal hemagglutinin- or whole virus-specific T cell lines and one of four T cell clones. This difference could not be accounted for simply by the ability of infectious virus to overcome a defect in antigen uptake by the M1-DR1 cells, in that direct studies of endocytosis showed that the M1 cells were more efficient than human B cells in the internalization of exogenous protein. These data suggested that the M1 cells were unable to present exogenous antigens but were capable of loading major histocompatibility complex (MHC) class II molecules with peptides derived from endogenous antigens. To investigate this further, the M1-DR1 cells were super-transfected with a cDNA encoding the p33 and p35 forms of the human invariant chain (Ii). Expression of the Ii chain was detected by intracytoplasmic staining of transfectants, and by metabolic labeling. Equimolar amounts of the p33 and p35 forms were detected, and the high level of p35 Ii was reflected by extensive retention of Ii protein in the endoplasmic reticulum. Addition of the Ii chain led to no recovery of presentation of intact antigens with DR1, but inhibited the presentation of live virus. These data indicate that MHC class II molecules in the M1-DR1 cells can be loaded with peptides derived from endogenous proteins, possibly in the biosynthetic pathway, and that the Ii chain has a role in limiting this route of class II antigen presentation.

Antigen Presentation↗
PubMed1994

The bio-logical role of invariant chain (Ii) in MHC class II antigen presentation.

Foreign antigens are internalized by antigen presenting cells by endocytosis and processed to peptides. To enable presentation of antigenic peptides by MHC class II molecules, these molecules have to be sorted to endosomal compartments where they can meet and bind the peptides. Invariant chain is complexed with MHC class II molecules and contains sorting signals responsible for MHC class II accumulation in endosomes. Invariant chain also has several other features contributing to the immune system's specific combat against invaders.

Animals↗
PubMed1994

Targeting of membrane proteins to endosomes and lysosomes.

The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.

Journal Article↗
PubMed1994

An LI and ML motif in the cytoplasmic tail of the MHC-associated invariant chain mediate rapid internalization.

Invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Two regions of the 30 residue cytoplasmic tail of Ii contain sorting information able to direct Ii to the endocytic pathway. The full-length cytoplasmic tail of Ii and the two tail regions were fused to neuraminidase (NA) forming chimeric proteins (INA). Ii is known to form trimers and when INA was transfected into COS cells it assembled as a tetramer like NA. The INA molecules were targeted to the endosomal pathway and cotransfection with Ii showed that both molecules appeared in the same vesicles. By labelling the INA fusion proteins with iodinated antibody it was found that molecules with either endocytosis signal were expressed at the plasma membrane and internalized rapidly. Point mutations revealed that an LI motif within the first region of the cytoplasmic tail and an ML motif in the second region were essential for efficient internalization. The region containing the LI motif is required for Ii to induce large endosomes but a functional LI internalization motif was not fundamental for this property. The cytoplasmic tail of Ii is essential for efficient targeting of the class II molecules to endosomes and the dual LI and ML motif may thus be responsible for directing these molecules to the endosomal pathway, possibly via the plasma membrane.

Amino Acid Sequence↗
PubMed1993

Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization.

Class II molecules of the major histocompatibility complex (MHC) bind peptides derived from protein antigens delivered into endocytic compartments and present these peptides to CD4+ T cells. The precursors to functional MHC class II molecules loaded with peptides are complexes of the invariant chain associated with class II alpha beta heterodimers. Targeting of newly synthesized MHC class II molecules to endosomes is mediated by the invariant chain, but the intracellular transport route is not known. This study demonstrates that in a human B-cell line a large population of MHC class II-invariant chain complexes reaches endosomes by rapid internalization from the cell surface. Quantitation of cell surface MHC class II-invariant chain complexes and of their surface half-life revealed that 3000 complexes internalized per minute into endosomes. This highly efficient endocytosis was mediated by the cytoplasmic tail of the invariant chain. After internalization, the invariant chain dissociated from the MHC class II-invariant chain complexes. This pathway may represent an important mechanism for loading class II molecules with immunogenic peptides from several endocytic compartments.

B-Lymphocytes↗
PubMed1993

Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.

Amino Acid Sequence↗
PubMed1993

Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

Animals↗