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Biomedical subjects

B Schraven

Publications and source records attributed to B Schraven.

At least 37 records · Page 2Linked to original sources
PubMed1999

Biochemical and functional analysis of mice deficient in expression of the CD45-associated phosphoprotein LPAP.

The role of the CD45-associated phosphoprotein (LPAP / CD45-AP) during an immune response remains unclear. To understand better the function of LPAP we generated LPAP-deficient mice by disrupting exon 2 of the LPAP gene. LPAP-null mice were healthy and did not show gross abnormalities compared to their wild-type littermates. However, immunofluorescence analysis of T and B lymphocytes revealed a reduced expression of CD45, which did not affect a particular subpopulation. In contrast to a recent report (Matsuda et al., J. Exp. Med. 1998. 187: 1863 - 1870) we neither observed significant alterations of the assembly of the CD45 / lck-complex nor of polyclonal T-cell responses. However, lymphnodes from LPAP-null mice showed increased cellularity, which could indicate that expression of LPAP might be required to prevent expansion of lymphocytes in particular lymphatic organs rather than potentiating immune responses.

Animals↗
PubMed1999

Coupling the TCR to downstream signalling pathways: the role of cytoplasmic and transmembrane adaptor proteins.

Engagement of the T-cell receptor (TCR) complex initiates a cascade of intracellular events ultimately leading to T-cell proliferation and differentiation. One of the first detectable consequences of TCR triggering is the activation of cytoplasmic protein kinases which, through phosphorylation of specific substrates, couple the TCR to downstream signalling cascades. Although it is well established that activation of the Ras- and the calcium-dependent calcineurin pathway is required for the achievement of T-cell activation, the precise mechanism as to how the TCR is connected to these intracellular effector molecules is still unclear. Major progress has been made in this regard with the molecular characterization of novel cytoplasmic and transmembrane molecules called adaptor proteins which integrate TCR-mediated signals at the intracellular level thus allowing fine tuning of T-cell responses.

Animals↗
PubMed1999

Expression of the membrane protein tyrosine phosphatase CD148 in human tissues.

CD148, a receptor-like protein tyrosine phosphatase also known as HPTP-eta/DEP-1, is involved in signal transduction in leucocytes and is thought to contribute to mechanisms of cellular differentiation. We have investigated the in situ expression of CD148 in various fresh-frozen tissues by immunohistology and analyzed its expression on subpopulations of activated peripheral blood leucocytes by flow cytometry. In lymphoid organs, CD148 was found to be widely expressed on B and T cells, granulocytes, macrophages, certain dendritic cells as well as mature thymocytes. The cellular level of CD148 was increased after in vitro activation of peripheral blood leucocytes. Comparative analysis of tissue samples from normal gut and from patients with active Crohn's disease showed that leucocytes expressing CD148 are significantly upregulated in inflamed tissues and that a subset of these cells co-express the activation marker CD25. In non-lymphoid tissues, CD148 was found to be present on many epithelial cell types with glandular and/or endocrine differentiation as well as on fibrocytes, melanocytes and Schwann cells. CD148 expression was maintained also in malignant counterparts of such tissues. However, a marked loss of CD148 immunoreactivity was apparent in some of the investigated high-grade carcinomas. In summary, our results confirm a role of CD148 as a leucocyte activation marker. Among non-hematopoietic cells, CD148 is expressed by characteristic types of epithelial and non-epithelial cells. Downregulation of CD148 might promote dedifferentiation and autonomous growth of such cells in malignant tumors.

Adenocarcinoma↗
PubMed1998

Molecular interaction between the Fyn-associated protein SKAP55 and the SLP-76-associated phosphoprotein SLAP-130.

It has previously been reported that in resting T-lymphocytes the protein tyrosine kinase p59 constitutively co-precipitates with four phosphoproteins of 43, 55, 85, and 120 kDa, respectively. We have recently cloned the 55-kDa protein that was termed Src kinase-associated phosphoprotein of 55 kDa (SKAP55). Here we demonstrate that the recently characterized SH2-domain-containing leukocyte protein 76-associated phosphoprotein of 130 kDa (SLAP-130) is one of the components of the Fyn complex and that it also co-precipitates with SKAP55 in human T-cells. We establish that SKAP55 and SLAP-130 associate with each other when both molecules are co-expressed in COS cells. By co-transfection of truncated mutants of SKAP55 and SLAP-130 as well as by using the two-hybrid selection system, we further demonstrate that the association between SLAP-130 and SKAP55 is direct and involves the Src homology 3 domain of SKAP55 and the proline-rich sequence of SLAP-130.

Adaptor Proteins, Signal Transducing↗
PubMed1998

SKAP-HOM, a novel adaptor protein homologous to the FYN-associated protein SKAP55.

A recombinant GST-Fyn-SH2 domain was used to purify proteins from lysates of pervanadate treated EL4 cells. N-terminal sequencing and molecular cloning of one of the purified polypeptides resulted in the identification of a novel adaptor protein that shares strong structural homology to the recently cloned Fyn-associated adaptor protein SKAP55. This protein was termed SKAP-HOM (SKAP55 homologue). Despite their striking homology, SKAP55 and SKAP-HOM have distinct characteristics. Thus, unlike SKAP55, which is exclusively expressed in T lymphocytes, SKAP-HOM expression is ubiquitous. Furthermore, while SKAP55 is constitutively tyrosine phosphorylated in resting human T cells, SKAP-HOM is expressed as a non-phosphorylated protein in the absence of external stimulus but becomes phosphorylated following T cell activation. In addition, SKAP-HOM does not associate with p59fyn in T cells although it represents a specific substrate for the kinase in COS cells. Finally, we demonstrate that, as previously shown for SKAP55, SKAP-HOM interacts with the recently identified polypeptide SLAP-130.

Amino Acid Sequence↗
PubMed1998

T cell receptor (TCR) interacting molecule (TRIM), a novel disulfide-linked dimer associated with the TCR-CD3-zeta complex, recruits intracellular signaling proteins to the plasma membrane.

The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor-bound protein 2 (Grb2), phospholipase Cgamma1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)- CD3-zeta complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR-CD3-zeta complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19-amino acid transmembrane region, and a 159-amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain-mediated protein-protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Biochemical analysis of the CD45-p56(lck) complex in Jurkat T cells lacking expression of lymphocyte phosphatase-associated phosphoprotein.

In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1998

Serum affinity chromatography for the detection of IgG alloantibodies in a patient with high-titer IgM cold agglutinins.

BACKGROUND AND OBJECTIVES: High-titer cold autoagglutinins (CA) interfere with the detection of alloantibodies against red-blood-cell antigens. The diagnostic procedure is extremely difficult, especially under pressure of time. It was the purpose of the study to find out whether quantitative IgG purification from serum or plasma by affinity chromatography facilitates the detection of IgG alloantibodies in the presence of high-titer IgM CA. MATERIALS AND METHODS: Serum: IgM-chi CA, 0 degrees C titer = 65,000; five anti-IgG reactive alloantibodies. Affinity chromatography: HiTrap protein G 1-ml affinity column (Pharmacia Biotech, Uppsala, Sweden). RESULTS: All IgG alloantibodies were detectable in the eluate after affinity chromatography without interference of IgM CA. CONCLUSION: IgG alloantibodies in the presence of high-titer IgM CA can be easily and rapidly detected under routine conditions by quantitative IgG purification from serum or plasma by affinity chromatography.

Anemia, Hemolytic, Autoimmune↗
PubMed1997

The receptor function of CD2 in human CD2 transgenic mice is based on highly conserved associations with signal transduction molecules.

The activation of human T cells via CD2 in response to mitogenic monoclonal antibodies (mAbs) typically requires that one mAb is specific for an epitope within the N-terminal Ig domain of CD2 and the other for a partially hidden epitope. We have examined the proliferative response of human T cells and human CD2 (huCD2) transgenic murine T cells to two novel CD2 monoclonal antibodies, AICD2.M1 and AICD2.M2, and have partially mapped the epitopes of these and other mitogenic CD2-specific monoclonal antibodies by way of recognition of CD2:CD58 chimeric proteins possessing either the N-terminal or the membrane proximal immunoglobulin domains of CD2. To understand the molecular basis of proliferation in huCD2 transgenic murine T cells, the interactions of huCD2 with signaling proteins in murine T cells were analyzed. The transgenic huCD2 molecule was found to interact with the murine tyrosine kinases p56lck and p59fyn and the CD3-epsilon and zeta chains of the TCR/CD3 signaling complex and to coimmunoprecipitate tyrosine phosphatase activity. These molecular associations resemble the situation in human T cells and suggest that human CD2 couples to the same signal transduction pathways in humans and transgenic mice.

Animals↗
PubMed1997

Molecular cloning of SKAP55, a novel protein that associates with the protein tyrosine kinase p59fyn in human T-lymphocytes.

In human T-lymphocytes the Src family protein tyrosine kinase p59(fyn) associates with three phosphoproteins of 43, 55, and 85 kDa (pp43, pp55, and pp85). Employing a GST-Fyn-Src homology 2 (SH2) domain fusion protein pp55 was purified from lysates of Jurkat T-cells. Molecular cloning of the pp55 cDNA reveals that the pp55 gene codes for a so far nondescribed polypeptide of 359 amino acids that comprises a pleckstrin homology domain, a C-terminal SH3 domain, as well as several potential tyrosine phosphorylation sites, among which one fulfills the criteria to bind Src-like SH2 domains with high affinity. Consistent with this observation, pp55 selectively binds to isolated SH2 domains of Lck, Lyn, Src, and Fyn but not to the SH2 domains of ZAP70, Syk, Shc, SLP-76, Grb2, phosphatidylinositol 3-kinase, and c-abl in vitro. Based on these properties the protein was termed SKAP55 (src kinase-associated phosphoprotein of 55 kDa). Northern blot analysis shows that SKAP55 mRNA is preferentially expressed in lymphatic tissues. SKAP55 is detected in resting human T-lymphocytes as a constitutively tyrosine phosphorylated protein that selectively interacts with p59(fyn). These data suggest that SKAP55 represents a novel adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes.

Amino Acid Sequence↗
PubMed1997

Identification of IgG alloantibodies in patients with high-titer IgM cold agglutinins by serum/plasma affinity chromatography.

The detection of IgG alloantibodies in the presence of high-titer cold autoagglutinins (CAs) can be extremely difficult, especially under pressure of time when transfusion of red blood cells is urgently needed. Here we demonstrate that IgG alloantibodies in the presence of high-titer IgM CAs can be easily detected by quantitative IgG purification from serum or plasma by affinity chromatography. In comparison with the routinely used methods for IgG alloantibody identification, affinity chromatography shows better or identical results and is the method leading to results most rapidly.

Agglutinins↗
PubMed1996

Sequence, genomic organization, and chromosomal localization of the human LPAP (PTPRCAP) and mouse CD45-AP/LSM-1 genes.

Human LPAP (lymphocyte phosphatase associated phosphoprotein) and its mouse homologue, CD45-AP (CD45 associated protein) or LSM-1, represent 32- and 30-kDa transmembrane phosphoproteins that noncovalently associate with the tyrosine phosphatase CD45, a key molecule involved in T- and B-lymphocyte activation. Here we report the isolation and sequencing of genomic clones of the human and mouse genes. LPAP (HGMW-approved symbol PTPRCAP) maps to human chromosome 11q13, distal to the BCL-1 breakpoint, and mouse CD45-AP/LSM-1 maps to Chromosome 19B. Both genes span 3 kb and consist of two exons that are separated by a 1.2-kb intron. The promoter regions do not contain TATA boxes, but possess consensus transcriptional initiator sequences that have also been described for other TATA-less genes. The genomic sequences also provide a genetic basis for two different cDNAs (termed CD45-AP and LSM-1, respectively) that have been described in the mouse system.

Animals↗
PubMed1996

Differential interaction of the CD2 extracellular and intracellular domains with the tyrosine phosphatase CD45 and the zeta chain of the TCR/CD3/zeta complex.

T cell activation via CD2 requires interaction of CD2 with several signaling molecules. To investigate the structural requirements for an association of CD2 with the tyrosine phosphatase CD45 and the zeta chain of the T cell receptor (TCR)/CD3/zeta complex, we have expressed in mouse EL4 T cells a series of human CD2 chimeric and mutant proteins. Chimeric proteins in which the CD2 transmembrane and/or cytoplasmic domains were deleted or exchanged with analogous regions of CD4, CD28 or CD58 retained association with high levels of murine CD45 phosphatase activity, suggesting that the CD2 extracellular domain largely controls interaction with CD45. To a lesser extent, the cytoplasmic domain of CD2 was also shown to interact with CD45, as demonstrated by an increase in co-immunoprecipitated phosphatase activity observed following replacement of the CD58 cytoplasmic domain with that of CD2. In contrast, the cytoplasmic domain of CD2 was found to be responsible for the majority of CD2 interaction with the zeta chain of the TCR/CD3/zeta complex. Deletion of the CD2 cytoplasmic domain, excluding the first three amino acids, removed virtually all CD2 associated zeta chain and approximately sevenfold higher levels of zeta chain were found in association with a CD58/58/2 chimera than with control human CD58 wild type. This study suggests that the CD2 extracellular and intracellular domains are differentially involved in regulating T cell activation through interaction with the tyrosine phosphatase CD45 and the zeta chain of the TCR/CD3/zeta complex.

Animals↗
PubMed1995

Identification of the sites of interaction between lymphocyte phosphatase-associated phosphoprotein (LPAP) and CD45.

Human lymphocyte phosphatase-associated phospho-protein (LPAP) is a phosphoprotein of unknown function that noncovalently associates with CD45 in lymphocytes. In CD45-deficient human T cells, LPAP protein is synthesized at normal levels but is more rapidly degraded than in wild-type cells. Expression of CD45 cDNA rescues LPAP protein expression. This strongly suggests that LPAP is protected from degradation through its interaction with CD45. We have mapped the sites of interaction between LPAP and CD45 employing chimeric CD45 molecules and LPAP deletion mutants. Our data demonstrate that the interaction between LPAP and CD45 is mediated via the transmembrane regions of both molecules. In addition, the intracytoplasmic amino acids adjacent to the transmembrane region of LPAP may influence its binding to CD45.

Amino Acid Sequence↗
PubMed1995

APO-1(CD95)-mediated apoptosis in Jurkat cells does not involve src kinases or CD45.

Tyrosine phosphorylation has been reported to be an early event required for APO-1/Fas(CD95) signalling in lymphocytes [Eischen, C.M., Dick, C.J. and Leibson, P.J. (1994) J. Immunol. 153, 1947-1954]. We have compared two mutant Jurkat cells, one largely deficient in expression of CD-45 (J45.01) and a second one deficient in expression of p56lck (JCaM1.6) with wild type Jurkat cells for their ability to undergo APO-1-induced apoptosis. No significant difference was observed among the three cell lines. In the mutant Jurkat cells APO-1 triggering did not result in increased tyrosine phosphorylation of cytosolic proteins. Furthermore, herbimycin A did not inhibit but rather augmented apoptosis at concentrations which effectively degraded the src related kinases lck and fyn. The data suggest that APO-1-mediated signalling is independent from src kinases and CD45.

Antigens, Surface↗
PubMed1995

Human T lymphocyte activation induces tyrosine phosphorylation of alpha-tubulin and its association with the SH2 domain of the p59fyn protein tyrosine kinase.

A glutathione-S-transferase-src-homology domain 2 (GST-SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, alpha- and beta-tubulin were identified by N-terminal sequencing. Further analysis established that alpha-tubulin exists as a tyrosine-phosphorylated protein in Jurkat cells, where it interacts with p59fyn, but not with p56lck. By contrast, in untransformed resting human T lymphocytes alpha-tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine-phosphorylated alpha-tubulin that is able to interact with the fyn-SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.

Amino Acid Sequence↗