Ephrin-B3 binds specifically to B lymphocytes in blood and induces migration.

Eph receptors and ephrin ligands have been shown to be differentially expressed on leucocytes. Here, we show that one member of the ephrin-B subfamily of ephrins, ephrin-B3, specifically binds to B lymphocytes in blood. No binding was observed to T lymphocytes or monocytes. The ephrin-B3 binding receptor on B lymphocytes is so far not identified, but our results here indicate that ephrin-B3 binds to a protein not belonging to the Eph receptor family. Recently, we have shown that ephrin-B3 binds to a sulphated cell surface receptor on HEK293T cells and that this binding can be blocked with heparin. Ephrin-B3 binding to B lymphocytes is partially affected by heparin, and a basic amino acid in the extracellular juxtamembrane region, Arg-188, is here shown to be involved in this binding. The functional consequence of ephrin-B3 binding to B lymphocytes is induced migration, in particular of the memory cells. To conclude, ephrin-B3 binds to B lymphocytes, most likely via a non-classical receptor, and induces migration of the memory B cell subpopulation.

Activation of EphA receptors on CD4+CD45RO+ memory cells stimulates migration.

We have demonstrated previously that binding of ephrin-A1 to EphA receptors on human CD4(+) and CD8(+) T cells stimulates migration. Two EphA receptors have been reported in T cells: EphA1 at the protein level and EphA4 at the mRNA level. In this study, we wanted to investigate the expression profile of these receptors in T cell subpopulations and to test if expression differences would affect the potential of cells to migrate upon ephrin-A1 binding. We have generated an anti-EphA4 mAb for expression analysis. Our data show that functional EphA4 is expressed on the cell surface of CD4(+) and CD8(+) T cells. In addition, EphA4 receptor expression is induced after overnight incubation in serum-free medium, in particular, on CD4(+)CD45RO(+) T cells. Migration of CD4(+) T cells in response to ephrin-A1 is observed for memory cells (CD45RO(+)) and much weaker for naïve cells (CD45RA(+)). A signaling complex associated with the EphA4 receptor has also been isolated and includes EphA1, the Src family kinases Fyn and Lck, Slp76, and Vav1. To conclude, T cells express EphA1 and EphA4 receptors. Expression differences of EphA4 are observed in subpopulations of CD4(+) T cells. This is related to the cell migration potential after ephrin-A1 binding.

Characterization, expression and functional aspects of a novel protein tyrosine phosphatase epsilon isoform.

This report describes the identification and characterization of a novel cytoplasmic isoform of human protein tyrosine phosphatase epsilon (PTPepsilon). The novel isoform, denoted cyt-PTPepsilonPD1, displays only the N-terminal catalytic, active phosphatase domain 1 (PD1) which is common in all known PTPepsilon isoforms. In addition, it contains a unique 132-residue long C-terminal end with no known motifs or homology to other characterized proteins. RNAse protection assay on isolated leucocyte subpopulations and selected cell lines demonstrated highest expression of cyt-PTPepsilonPD1 in monocytes. The mRNA-encoding cyt-PTPepsilonPD1 is detected as distinct transcript(s) by Northern blot analysis and is a result of alternative splicing. cyt-PTPepsilonPD1 shows similar cellular localization in transfected cells, both in the cytoplasm and nucleus, as has been previously described for cytoplasmic PTPepsilon isoform. Our previous data suggest that the expression of cytoplasmic PTPepsilon inhibits the mitogen-activated protein kinase cascade through the extracellular signal-regulated kinase 1 and 2 pathway. A similar functional role is also presented here for cyt-PTPepsilonPD1, supporting our previous data suggesting that the catalytic first PD of PTPepsilon is responsible for this inhibition.

A signal sequence trap based on cell enrichment using anti-CD19 antibody coated magnetic beads.

Precursors of many secreted and cell surface proteins contain NH2-terminal signal sequences that lead proteins into the endoplasmic reticulum and to the cell surface. Methods have been developed to clone and identify such proteins by trapping their NH2-terminal signal sequences, so called signal sequence traps. In this study we present an alternative and simplified signal sequence trap based on the combination of a novel vector construct expressing a cDNA library in fusion with a CD19 reporter gene, transfection in mammalian cells and selection of cells expressing trapped signal sequences using magnetic beads. Using this method we have isolated several known and novel factors with signal peptides.

Identification of nucleolar protein No55 as a tumour-associated autoantigen in patients with prostate cancer.

Four different genes were identified by immunoscreening of a cDNA expression library from the human prostate cancer cell line DU145 with allogeneic sera from four prostate cancer patients. A cDNA encoding the nucleolar protein No55 was further analysed and shown to be expressed at the mRNA level in several normal tissues, including ovaries, pancreas and prostate and in human prostate cancer cell lines PC-3, PC-3m and LNCaP. By reverse transcriptase/polymerase chain reaction, expression of No55 was several-fold higher in two out of nine prostate cancer primary tumours and two out of two metastatic lesions, compared to normal prostate tissue. Antibodies to No55 were detected in sera from seven out of 47 prostate cancer patients but not in sera from 20 healthy male controls. Sequence analysis of the No55 open reading frame from normal and tumour tissues revealed no tumour-specific mutations. The No55 gene was located to chromosome 17q21, a region reported to be partially deleted in prostate cancer. Considering the immunogenicity of the No55 protein in the tumour host, the expression profile and chromosomal localization of the corresponding gene, studies evaluating No55 as a potential antigen for immunological studies in prostate cancer may be warranted.

The C1orf9 gene encodes a putative transmembrane member of a novel protein family.

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.

A splice variant of human ephrin-A4 encodes a soluble molecule that is secreted by activated human B lymphocytes.

Ephrin-A4 is a ligand for the erythropoietin-producing hepatocellular (Eph) receptor family of tyrosine kinases. We have identified a secreted form of ephrin-A4, denoted ephrin-A4 (s), which is encoded by an alternatively spliced mRNA and is produced by in vivo activated B cells in tonsils. Blood B cells secrete ephrin-A4 (s) upon stimulation via the B-cell antigen receptor. A subpopulation of tonsil cells in the crypts with a dendritic cell phenotype was shown to express EphA2, an Eph receptor tyrosine kinase that was found to be capable of binding an ephrin-A4 immunoglobulin chimeric protein. We conclude that ephrin-A4 (s) may play a role in the interaction between activated B lymphocytes and dendritic cells in human tonsils. (Blood. 2000;95:221-230)

Identification and characterization of TESK2, a novel member of the LIMK/TESK family of protein kinases, predominantly expressed in testis.

In this study we present the cDNA sequence of a novel putative protein kinase, denoted TESK2. The open reading frame of TESK2 encodes a putative 555-amino-acid protein, including a protein kinase consensus sequence in the N-terminal half. The protein kinase domain of TESK2 is structurally similar to the kinase domain of the protein serine/threonine kinase TESK1 (64% identity) and to those of the LIMK1 and LIMK2 kinases (42 and 39% identity, respectively). TESK2, together with TESK1, constitutes a second subgroup of the LIMK/TESK family of protein kinases, as revealed by phylogenetic analysis of the protein kinase domains. Chromosomal localization of human TESK2 was assigned to 1p32. Expression analysis of human TESK2 revealed a single mRNA species of 3.0 kb predominantly expressed in testis and prostate and low expression in most other tissues examined. Rat testicles expressed a single species of TESK2 mRNA of approximately 3.5 kb. However, the transcript was first detectable in rat testis after day 30 of postnatal development and was predominantly expressed in round spermatids. These observations suggest that TESK2 plays an important role in spermatogenesis.

Cloning, chromosal mapping, and tissue expression of the gene encoding the human Eph-family kinase ligand ephrin-A2.

The Eph-related receptor tyrosine kinases constitute a large family of receptors with most members displaying specific expression patterns in the developing embryo. Ligands for Eph receptor tyrosine kinases, recently renamed ephrins, comprise a family of at least 8 membrane-bound members that display promiscuous binding to Eph receptors. Here we report the characterization of a human cDNA clone with high homology to the gene encoding the murine ephrin-A2 ligand. The human gene encodes a single 2.4-kb mRNA with a restricted and developmentally-regulated tissue distribution pattern. In the fetus, ephrin-A2 mRNA is expressed in brain and intestine, whereas in the adult, high levels of ephrin-A2 mRNA are detectable in lung and intestine. Using PCR-based screening of genomic DNA from human x rodent hybrid cell lines, the gene encoding ephrin-A2 (EFNA2) was assigned to chromosome 19. Fluorescence in situ hybridization to metaphase chromosome preparations refined this localization to band p13.3.

Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites.

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.

Regulated expression of the Eph-related receptor tyrosine kinase Hek11 in early human B lymphopoiesis.

Members of the large Eph family of receptor tyrosine kinases (RTKs) display temporally and spatially restricted expression patterns during embryogenesis, suggesting a role in various developmental processes. We have begun to investigate the expression of members of this receptor family during human hematopoiesis, in particular B lymphopoiesis. Expression of Eph RTKs in cells of the B-lymphoid lineage was assessed by using degenerate oligonucleotide primers based on stretches of conserved nucleic acid sequences in members of the Eph family. First, the content of Eph-family RTKs was assessed in freshly sorted fetal bone marrow pro-B cells. This population was found to harbor transcripts of the Hek8 and Hek11 members of this gene family. Subsequent analysis of expression of these genes in B cells representing various differentiation and ontogenic stages showed that the Hek8 transcript is constitutively present in all fetal and adult B-lineage cells, with high levels of expression in peripheral blood B cells. In contrast, the Hek11 transcript was exclusively found in fetal bone marrow pro-B cells and pre-B cells, but not in more mature fetal B-lineage cells. All adult B-lineage cells, from early pro-B cells to end-stage plasma cells, lacked Hek11 transcripts. The developmentally regulated expression of Hek11 during fetal B lymphopoiesis suggests a role for this gene in pre/pro-B cell expansion and/or differentiation and defines a difference in progenitor B cell populations isolated from fetal versus adult human bone marrow.

Characterization, expression and chromosomal localization of a human gene homologous to the mouse Lsc oncogene, with strongest expression in hematopoetic tissues.

A human cDNA clone, denoted sub1.5, was isolated from cDNA library generated from human T cells. The sub1.5 cDNA sequence was novel and was not identical to any known cDNA sequences in the GenBank. Recently, however, a mouse cDNA (Lsc) with high homology to sub1.5 was identified, indicating that the sub1.5 sequence may represent the human homologue of the mouse Lsc gene. The sub1.5 cDNA includes an open reading frame of 875 amino acids, predicting a protein with molecular weight of 97 kDa. Like Lsc, sub1.5 shows homology to the previous described oncogene Lbc, in particular to two functional domains in the Lbc protein; the Dbl proto-oncogene homology domain and the pleckstrin homology domain. Lsc is proposed to be an oncogene and is a member of a growing family of proteins that may function as activators of the Rho family GTPases. Members of the Rho family regulates the polymerization of actin to produce stress fibers. Activation of Rho GTPases by sub1.5 is also indicated by our studies, as stress fiber formation is observed in serum-starved stable NIH3T3 sub1.5 transfectants. Sub1.5 cDNA hybridizes to two major transcripts of 3.5 and 5 kb size and the strongest expression is seen in hematopoietic tissues like thymus, lymph nodes, peripheral blood leukocytes and spleen. We also show that both purified B and T cells express sub1.5. In addition, our data indicate that sub1.5 mRNA is abundantly expressed in CD34+ human progenitor cells. Fluorescent in situ hybridisation, using sub1.5 cDNA as a probe on human metaphases, shows that the sub1.5 gene is localized to chromosome 19q13.13.

A simple subtraction method for the isolation of cell-specific genes using magnetic monodisperse polymer particles.

In this study we present a simple subtraction method for the isolation of cell-type-specific genes using magnetic beads. Biotinylated first-strand cDNA is generated from one cell type and immobilized onto magnetic streptavidin beads. Poly A+ RNA, isolated from a different cell type by use of oligo-dT beads, is then hybridized to the immobilized cDNA. Beads with hybridized mRNA are subsequently removed from the solution by attraction to a magnet. The cell-specific mRNA, left in solution, is finally converted to a radiolabeled cDNA probe in order to screen cDNA libraries. In this study, we present an example of a successful subtraction strategy involving three cell types: the pre-B-cell line Reh; 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated Burkitt's lymphoma line Daudi; and TPA-stimulated T lymphocytes. This strategy was chosen due to our interest in gene products known to be expressed in TPA-stimulated B and T lymphocytes, but not in Reh cells. Several previously unknown genes were identified.

Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K.

In human cells at least 20 different proteins or groups of proteins have been identified that are associated with hnRNAs. These proteins (designated A1-U) are highly abundant in the nucleus. In this study, we present the sequence of a novel cDNA clone, sub2.3, isolated from a human lymphocyte cDNA library. The predicted amino acid sequence shows homology to repeated domains in the human hnRNA binding protein K (hnRNP K), which are believed to be of functional importance. hnRNP K is among the major oligo(rC/dC) binding proteins in vertebrate cells and we show here that the protein product of sub2.3 also binds to oligo(dC). This is shown by a novel approach where we demonstrated specific binding of in vitro translated sub2.3 protein to biotinylated oligo(dC) which was immobilized on magnetic streptavidin-coated Dynabeads. Moreover we found that the sub2.3 transcript is expressed in a tissue dependent manner with the highest expression observed in several lymphoid tissues and skeletal muscle. The gene was also abundantly expressed in several lymphoid cell lines and the hepatoma cell line HepG2 while a low expression was observed in the HL60 myeloid cell line and in the HeLa cervical carcinoma cell line. In conclusion, this study presents the cDNA sequence of a novel transcript which shows tissue specific expression and encodes a protein with oligo(dC) binding specificity in vitro.

Cell-specific expression of human beta-galactoside alpha 2,6-sialyltransferase transcripts differing in the 5' untranslated region.

In humans, two cDNAs have been isolated encoding beta-galactoside alpha 2,6-sialyltransferase, differing only in part of the 5' untranslated region. Primer extension data show that the two cDNAs are near full-length clones. RNase protection analysis of different cell types showed that the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a B-cell library resided only in mature B cells. In contrast, the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a placenta library was found in all cells tested. Our results also indicate the existence of a third alpha 2,6-sialyltransferase transcript in the hepatoma cell line HepG2. Mature B cells were found to express high amounts of alpha 2,6-sialyltransferase mRNA, compared to other cell types tested, as shown by Northern blot analysis. Moreover there was an increased expression of beta-galactoside alpha 2,6-sialyltransferase mRNA in activated B cells compared to resting B cells. In vitro transcription and translation of the cDNAs resulted in a protein of 45 kDa, but the transcripts were translated with different efficiency, suggesting a role for the 5' untranslated region in regulation of translation. We have also made an alpha 2,6-sialyltransferase construct lacking the specific 5' regions of the two cDNAs. A transcript generated from this construct was translated more efficiently in vitro than the two alpha 2,6-sialyltransferase cDNAs.