Showing papers by "Takashi Saito published in 1994"

Preferential usage of the Fc receptor gamma chain in the T cell antigen receptor complex by gamma/delta T cells localized in epithelia.

Abstract: zeta and eta chains of the T cell antigen receptor (TCR) complex and the gamma chain of Fc receptors (FcR gamma) constitute a family of proteins important for the expression of, and signal transduction through, these receptors in hematopoietic cells. In zeta-deficient mice, TCR expression was reduced in most T cells. By contrast, CD8 alpha alpha + TCR-gamma/delta + intestinal intraepithelial lymphocytes in these mice expressed a normal level of TCR. Biochemical analysis of the TCR complex in these cells from zeta-deficient as well as normal mice revealed the predominant usage of FcR gamma. Furthermore, gamma/delta + T cells in epithelia of the skin and female reproductive organs from zeta-deficient mice also showed relatively high TCR expression, indicating the usage of FcR gamma. These observations demonstrate the preferential usage of FcR gamma by gamma/delta + T cells localized in epithelia of normal mice.

High frequency of cancer patients with abnormal assembly of the T cell receptor-CD3 complex in peripheral blood T lymphocytes.

Abstract: Structural abnormality of T cell receptor (TCR)-CD3 complex on the cell surface was investigated in peripheral blood lymphocytes (PBL) from 55 cancer patients In 24 of the 68 tests done on these patients, the CD3C chain was not detected by immunoprecipitation with anti-CD3ζ monoclonal antibody (mAb), but was observed with anti-CD3ζ mAb, suggesting that a high frequency of cancer patients possesses abnormal T cell receptor (TCR) complex in PBL On the other hand, the total ζ chain was missing in several advanced cases During follow-up of several patients, the ζ chain became undetectable after two or three months of cancer progression It appears that immunosuppressive status can be monitored by analyzing the TCR-CD3 complex on the cell surface of PBL

Purification of glutathione S-transferase fusion proteins as a non-degraded form by using a protease-negative E. coli strain, AD202.

Abstract: Recombinant proteins prepared by genetic engineering are utilized for various purposes in molecular biology such as production of specific antibodies or investigation of the mechanism of protein—DNA or protein-protein interactions (1). There are several methods commonly used to produce recombinant proteins in bacteria or insect cells (2). Expression of a recombinant protein in E. coli as a fusion protein with glutathione S-transferase (GST) is one of the most popular and easiest methods (3). However, we often encounter the serious problems that some fusion proteins are rapidly degraded or cannot be solubilized in bacterial cells. To prepare a polyclonal antibody against the 7 chain of the high affinity IgE receptor (FCCRIY), we constructed GST-FceRI-y, which was composed of the GST gene joined to the cytoplasmic portion of the FceRty cDNA. In initial experiments, we utilized two commonly used bacteria strains, DH5a and TGI, for production of the fusion protein. Although the GST-fusion protein was induced with isopropyl-/3-D-thiogalactopyranoside and solubilized in the supernatant of the bacterial lysate after sonication, the fusion protein was rapidly cleaved at the fusion joint between the GST and FwRI-y during purification (Fig l.A). This cleavage could not be prevented by the addition of several available protease inhibitors such as phenylmethyl sulfonyl fluoride, aprotinin, leupeptin, bestatin and ethylenediaminetetraacetic acid (data not shown). Several trials for alternative methods such as shortening the induction time, changing the incubation temperature, or lysis with freezing and thawing method instead of sonication did not improve the results at all (data not shown). To overcome this problem, we then used several protease negative E.coli strains (Table 1). In one of these strains, AD202, which is defective in ompT encoding an outer membrane-associated protease (4), the fusion protein was not cleaved at all and recovered from bacterial lysate with an expected molecular size (Fig. 1A). The prevention of degradation was observed not only with this construct but also with other GST-fusion proteins including GST-CD3e (Fig. IB). It is noteworthy that a fusion protein, GST-CD3f, which could not be detected in DH5a, could be induced and purified by using AD202 (Fig. 1C). These results suggest that an extremely unstable fusion protein such as GSTCDS f can be stabilized in AD202. We next examined the efficiency of this strain by using a larger fusion protein construct. A 75 kD GST-CTBPl, which contains a coding frame of a singlestranded DNA and RNA binding protein of the yeast, was purified efficiently in AD202, whereas most of the products were degraded in DH5a (Fig. ID). This suggests that AD202 is also effective for fusion proteins as large as 75 kD and for those encoding nucleic acid binding proteins. From these observations, we conclude that AD202 is one of the most appropriate strains for the purpose of producing GST-fusion proteins in E. coli.

Different cytoplasmic structure of the CD3ζ family dimer modulates the activation signal and function of T cells

Abstract: The TCR complex transduces the antigen recognition signal through common activation motifs present in both CD3 gamma delta epsilon chains and zeta dimers within the complex. We have investigated functional roles of the cytoplasmic domain in zeta and CD3 gamma delta epsilon for T cell activation in early and late responses by comparing the signaling capability of the TCR complexes containing mutant zeta lacking some or all motifs, or eta chain, another zeta family molecule. The results with the mutant zeta lacking all motifs indicated that CD3 gamma delta epsilon can transduce signals to cause early activation events and production of IL-2 upon antigen stimulation in the absence of zeta motifs. However, any one of the zeta motifs was required to respond to Thy-1 stimulation and this requirement cannot be replaced by other CD3 chains. Such zeta motif-dependent responses were also observed in tyrosine phosphorylation of a 90 kDa protein upon TCR stimulation. Furthermore, we found that the C-terminal unique region of the eta chain exhibits inhibitory function in phosphorylation and Ca2+ response upon TCR stimulation as well as IL-2 production upon Thy-1 stimulation. Collectively, the present analyses suggest that two types of signals are induced through the TCR-CD3 complex: (i) the common motif-dependent signals which are mediated equally through zeta dimers and CD3 gamma delta epsilon, and (ii) zeta specific motif-dependent signals. Differences in the cytoplasmic domain of zeta family molecules may modulate the cooperation of these two signals, resulting in alteration of T cell functions.

Transition from TCR-beta dimer to TCR-alpha beta-expressing cells by introduction of an alpha-chain in an immature thymocyte cell line.

Abstract: The molecular basis for the surface expression of TCR-beta chain in the absence of association with TCR-alpha, -gamma, or -delta chain by an immature thymocyte cell line was investigated. The TCR-beta chain expressed by this cell line was not encoded by any unique DNA sequence, nor was it inserted into the membrane via a glycosyl-phosphatidylinositol linkage. Transfection of two other beta-chains derived from mature T cell clones resulted in the surface expression of dimers of the transfected beta-chains in both cases. Immunoprecipitation of the beta-dimer-CD3 complex demonstrated that the association of the beta-dimer with the CD3 complex, especially the CD3 zeta chain, was so weak that they dissociated under the detergent conditions in which the TCR-CD3 complex of mature T cells is kept intact. Transfection of TCR-alpha chain resulted in the expression of a TCR-alpha beta-CD3 complex and the disappearance of beta-dimers. In accordance with the changes in TCR complex components, the association between TCR-alpha beta and CD3 complex became stable and the cells transduced signals more efficiently. The results demonstrate that the expression of TCR-beta as part of an incomplete TCR-CD3 complex is developmentally regulated and the expression of TCR-alpha chain results in normal configuration and function of TCR complex.

Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line.

Abstract: Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.