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P Angelisová

Publications and source records attributed to P Angelisová.

At least 19 recordsLinked to original sources
PubMed2001

Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Actins↗
PubMed2001

A proteolytically truncated form of free CD18, the common chain of leukocyte integrins, as a novel marker of activated myeloid cells.

An unusual CD18 monoclonal antibody (mAb) MEM-148 binds, in contrast to standard CD18 mAbs, specifically to peripheral blood monocytes and neutrophils activated by various stimuli such as phorbol myristate acetate, opsonized zymosan, heat-aggregated immunoglobulin, and (after priming with lipopolysaccharide, tumor necrosis factor, or granulocyte-macrophage colony-stimulating factor) also by formyl-methionyl-leucyl-phenylalanine. In addition, in vivo activated neutrophils obtained from urine of patients following recent prostatectomy were also strongly positive for MEM-148. On the activated myeloid cells the mAb recognized a 65- to 70-kd protein identified immunochemically and by mass spectrometric peptide sequencing as a membrane-anchored fragment of CD18 (the common chain of leukocyte integrins) produced by proteolytic cleavage. The CD18 fragment originated mainly from integrin molecules stored intracellularly in resting cells, it was unassociated with CD11 chains, and its formation was inhibited by several types of protease inhibitors. Thus, the 65- to 70-kd CD18 fragment represents a novel abundant activation marker of myeloid cells of so far unknown function but possibly involved in conformational changes in leukocyte integrin molecules resulting in increased affinity to their ligands.

Antibodies, Monoclonal↗
PubMed2001

A novel anti-CD18 mAb recognizes an activation-related epitope and induces a high-affinity conformation in leukocyte integrins.

Monoclonal antibody MEM-148 was previously shown to recognize CD18 chains in a free form unassociated within leukocyte integrin heterodimers, but yet it is paradoxically able to induce a high-affinity conformation in the native, cell surface expressed LFA-1 molecules. Our results based on kinetics of binding, immunoprecipitation and cell-aggregation experiments demonstrate that the mAb does bind to and stabilizes a specific conformation of LFA-1 heterodimers apparently distinguished by an increased affinity to its cellular ligand(s). A similar high-affinity conformation of LFA-1, in which the MEM-148 epitope becomes exposed, is induced also by a Mg2+/EDTA or low pH (5.5-6.5) treatments which may mimic physiologically relevant situations in normal or inflamed tissues. Thus, mAb MEM-148 is a novel valuable tool for detection and induction of specific conformations of human leukocyte integrins.

Animals↗
PubMed2000

Human leukocytes contain a large pool of free forms of CD18.

Monoclonal antibodies to CD18, the common chain of leukocyte integrins, recognize in various types of human lymphoid and myeloid cells under the conditions of nonreducing Western blotting three species of CD18 of mol. wt. 96, 87, and 78 kDa, respectively. Using a unique monoclonal antibody MEM-148 reacts exclusively with free CD18 molecules, but not with leukocyte integrin heterodimers. We demonstrate that only the upper one (96 kDa) is present on the cell surface within the CD11/CD18 integrin heterodimers, while the lower ones (87 and 78 kDa) are found intracellularly as free molecules unassociated with CD11 chains or other molecules. These intracellular free CD18 chains may in part represent biosynthetic precursors; alternatively, these species may represent an intracellular source of the recently observed free, proteolytically truncated CD18 chains expressed on the surface of activated myeloid cells.

Antibodies, Monoclonal↗
PubMed2000

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

Adaptor Proteins, Signal Transducing↗
PubMed2000

CDw149 antibodies recognize a clustered subset of CD47 molecules associated with cytoplasmic signaling molecules.

One of the recently described antigens broadly expressed on human leukocytes is CDw149, which was defined at the 6th Human Leukocyte Differentiation Antigen (HLDA) Workshop by means of 2 monoclonal antibodies (mAbs). Molecular characterization of this antigen has been lacking. In the present study we demonstrate that these anti-CDw149 mAbs actually recognize a clustered subset of a well-defined membrane protein, CD47, also known as integrin-associated protein (IAP). This clustered subset is present on leukocytes but not erythrocytes. The anti-CDw149 mAbs bind with only low affinity to a monomeric (unclustered) subset of CD47 but with high avidity to the CD47 clusters. A fraction of CD47 is associated with large complexes containing cytoplasmic signaling molecules (Src family kinases and heterotrimeric G-proteins) similar to glycosphingolipid-enriched microdomains (GEMs), which may explain the previously described signaling capacity of CD47. The low-affinity anti-CD47 mAbs may be useful tools targeting specific receptor complexes involved in cell activation. Specific reactivity of low-affinity mAbs with clustered subsets of cell surface antigens may more generally explain the nature of poorly defined "activation forms" or activation neoepitopes described previously for several cell surface molecules.

Antibodies, Monoclonal↗
PubMed1999

GPI-microdomains: a role in signalling via immunoreceptors.

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids are assembled on the leukocyte surface within membrane microdomains, which also accommodate a set of cytoplasmic signalling molecules (Src family kinases, G-proteins, linker proteins). Recent results suggest that these membrane specializations mediate not only signal transduction via GPI-proteins and glycolipids but also play important roles in initiation of signalling via immunoreceptors.

Animals↗
PubMed1999

Characterization of the human leukocyte GPI-anchored glycoprotein CDw108 and its relation to other similar molecules.

The CDw108 glycoprotein is expressed on the surface of some leukemic cell lines, erythrocytes and on activated lymphocytes. Its surface expression is rapidly upregulated following various activating stimuli (PHA, PWM, Con A, PMA, anti-CD3) and subsequently gradually decreases. The molecule is anchored in the membrane via glycosylphosphatidylinositol (GPI) moiety, it has molecular mass of 75-80 kDa and pI of 5.0-5.5. Endoglycosidase F and H reduce its apparent size as determined by SDS PAGE by approx. 15 and 22 kDa, respectively. It is a component of large, detergent-resistant GPI-complexes associated with protein kinases. In addition to the previously described identity of CDw108 with the JMH blood group antigen, we demonstrate here its identity to the previously described glycoprotein recognized by monoclonal antibodies H105 and KS.2, and exclude its identity with another GPI-anchored glycoprotein of similar size, melanotransferrin (gp97).

Antigens, CD↗
PubMed1999

The nature of the subset of MHC class II molecules carrying the CDw78 epitopes.

A CDw78 mAb FN1 was shown to recognize DP and/or DR molecules under the conditions of Western blotting. DP molecules were specifically retarded on a column of the FN1 immunosorbent; binding of FITC-labeled FN1 to B cell lines was completely blocked by excess of mAb to DR/DP beta chains, partially by several mAb to DP and weakly by some mAb to DR. The binding of two other CDw78 mAb, FN4 and MR11, to the B cell surface was most strongly inhibited by excess of different mAb to DR. Kinetics of stable binding of the CDw78 mAb indicated that their monovalent binding is of low affinity and that the stable binding to the surface is due to bivalent binding to two spatially close MHC class II molecules. FN1-based immunosorbent effectively immunoisolated complexes of MHC class II proteins with several tetraspanin molecules from a mild detergent lysate of a B cell line. It is concluded that FN1 and most likely also the other two CDw78 mAb recognize with low affinity determinants on MHC class II molecules (DP or DR) and preferentially bind in a stable fashion to dimerized or aggregated MHC class II molecules. Such dimers or aggregates may either exist as preformed on the cell surface or may be gradually formed and stabilized by bivalent interaction with mAb. These structures may be related to the previously described 'superdimers' of MHC class II and/or 'MHC-tetraspanin complexes'. CDw78 mAb may be valuable tools targeting such aggregated fraction of MHC class II molecules which can exhibit important signaling and antigen-presenting properties.

Antibodies, Monoclonal↗
PubMed1998

Signal transduction in leucocytes via GPI-anchored proteins: an experimental artefact or an aspect of immunoreceptor function?

Membrane proteins anchored in the membrane via a glycolipid glycosylphosphatidylinositol (GPI) as well as some glycolipids are able to transduce signals and induce diverse functional responses in cells upon their cross-linking via antibodies or natural ligands. In some cases this signaling capacity seems to be due to associations of these molecules with specific transmembrane proteins. GPI-anchored proteins are components of membrane microdomains enriched in glycosphingolipids and cholesterol and devoid of most transmembrane proteins. These membrane specializations are relatively resistant to solubilization in solutions of some mild detergents at low temperatures. These 'GPI-microdomains' contain also cytoplasmic signaling molecules such as Src-family protein tyrosine kinases and trimeric G-proteins. Thus, at least some signaling elicited upon cross-linking of GPI-anchored proteins and glycolipids may be due to perturbation of the signaling molecules associated with these microdomains. It is suggested that these specialized areas of the membrane rich in signaling molecules interact with immunoreceptors (TCR, BCR, Fc receptors) cross-linked upon their interactions with ligands and importantly contribute to initiation of proximal phases of their signaling pathways.

Animals↗
PubMed1996

Association of the putative B-lymphocyte surface molecule B7.3 with a protein kinase activity.

Monoclonal antibody BB1 was previously shown to recognize the CD80 protein as well as a putative B7.3 molecule, both ligands of the important T-cell receptors CD28 and CTLA-4. We report that BB1 coprecipitated from detergent lysates of human B- and pre-B-cell lines a protein kinase activity, as detected by solid phase immunoprecipitation followed by in vitro kinase assay. As other monoclonal antibodies to CD80 did not co-precipitate any kinase activity, it seems likely that this protein kinase is associated with the so far poorly characterized putative B7.3 molecule.

Antibodies, Monoclonal↗
PubMed1996

Supramolecular complexes of MHC class I, MHC class II, CD20, and tetraspan molecules (CD53, CD81, and CD82) at the surface of a B cell line JY.

The results of previous biochemical studies indicated that a fraction of MHC class II proteins is associated with four proteins of the tetraspan family, CD37, CD53, CD81, and CD82, and possibly with other membrane components, at the surface of JY B lymphoma cells. In the present communication we used a biophysical technique, namely the flow cytometric energy transfer method, to demonstrate the proximity of these molecules at the surface of the cells. Significant energy transfer (and, therefore, proximity within the 2-10 nm range) was observed between fluorescently labeled mAbs to DR, DQ, and the tetraspan molecules CD53, CD81, and CD82. Moreover, two other B cell surface molecules, CD20 and MHC class I, were found to be close to each other and to MHC class II and the tetraspan proteins, based on the observed high energy transfer efficiencies between the relevant fluorescently labeled mAbs. The character of simultaneous energy transfer from CD20, CD53, CD81, and CD82 to DR suggests that all these molecules are in a single complex with the DR molecules (or a complex of several DR molecules) rather than that each of them is separately associated with different DR molecules. Based on these data and previous biochemical results, a model is proposed predicting that the B cell membrane contains multicomponent supramolecular complexes consisting of at least two MHC class I and at least one DR, DQ, CD20, CD53, CD81, and CD82 molecules. Closer analysis of the energy transfer efficiencies makes it possible to suggest mutual orientations of the components within the complex. Participation of other molecules, not examined in this study (CD19 and CD37), in these supramolecular structures cannot be ruled out. These large assemblies of multiple B cell surface molecules may play a role in signaling through MHC molecules and in Ag presentation to T cells.

Antibodies, Monoclonal↗
PubMed1994

Association of the GPI-anchored leucocyte surface glycoproteins with ganglioside GM3.

The major glycolipid co-immunopurifying with the glycosylphosphatidylinositol-anchored leucocyte surface glycoprotein CD59 from detergent lysates of human T cell lines HPB ALL, Jurkat and myeloid line HL-60 was identified as the glycosphingolipid GM3. Monoclonal antibodies to GM3 immunoprecipitated the same large detergent-resistant, protein-tyrosine kinase containing "GPI-complexes" as antibodies to several GPI-anchored proteins. Therefore GM3 is another component of these large membrane complexes potentially involved in signalling through GPI-anchored receptors or through some glycolipids.

Antibodies, Monoclonal↗
PubMed1994

Association of four antigens of the tetraspans family (CD37, CD53, TAPA-1, and R2/C33) with MHC class II glycoproteins.

Four of the tetraspans family antigens expressed in B cells, CD37, CD53, TAPA-1, and R2/C33, as well as at least two other molecules, CD19 and CD21, coprecipitate with DR antigens from mild detergent lysates of human B-cell lines and tonsillar B cells. Coprecipitation and preclearing experiments indicate the existence of large multicomponent complexes containing jointly the seven components, although some "incomplete" complexes lacking some of the components may also exist. The complexes contain only a relatively small fraction of the total cellular pool of relevant molecules. The existence of these "tetraspans-DR complexes" may be related to the previously reported antiproliferative and signaling effects of antibodies against most of their components.

Antibodies, Monoclonal↗
PubMed1994

Association of GPI-anchored glycoproteins with other components of the leucocyte membrane.

The leucocyte surface glycosylphosphatidylinositol (GPI)-anchored membrane proteins are localized within specific membrane microdomains which also contain specific (glyco)lipids and intracellular proteins including protein kinases. These "GPI-domains" are devoid of most abundant transmembrane proteins, but in T-cells they appear to contain small amounts of CD4 and CD8 and in B-cell lines, small amounts of CD10. The existence of these relatively detergent-resistant membrane microdomains explains the signal-transducing ability of GPI-anchored receptors. In addition to the "GPI-microdomains", several other types of analogous very large detergent-resistant complexes/domains appear to exist, such as those containing T-cell receptor, others containing CD45R molecules associated with a protein kinase, and still others composed mainly of several proteins of the tetraspan family. Therefore, we suggest that the leucocyte surface is a mosaic of microdomains of unique composition associated with specific signal-transducing molecules.

Antigens, CD↗