Life Sciences Research for Lifelong Health


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Title / Authors / Details Open Access Download

Analysis of peptide length preference of the rat MHC class Ia molecule RT1-A(u), by a modified random peptide library approach.
Stevens J, Wiesmüller KH, Butcher GW, Joly E

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

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International immunology, 12, 0953-8178, 83-9, 2000

PMID: 10607753

Open Access

Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells.
Oxley D, Bacic A

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, D-Manalpha(1-2)-D-Manalpha(1-6)-D-Manalpha(1-4)-D-GlcN -inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial beta(1-4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

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Proceedings of the National Academy of Sciences of the United States of America, 96, 0027-8424, 14246-51, 1999

PMID: 10588691

Activation and selection of NK cells via recognition of an allogeneic, non-classical MHC class I molecule, RT1-E.
Petersson E, Holmdahl R, Butcher GW, Hedlund G

Previous studies have established that NK cells express both inhibitory and activatory receptors. The inhibitory receptors have been shown to recognize major MHC class I molecules, but the physiological ligands for the activatory receptors have been only partly characterized. In this study we investigated whether NK cells could be activated by recognizing specific non-classical MHC class Ib molecules. NK cells from BN (RT1(n)) rats immunized in vivo with MHC-incompatible WF (RT1(u)) cells displayed cytolytic activity specific for product(s) of the MHC class Ib RT1-E(u) / C(u) region. These cells were shown to kill Rat2 fibroblast cells transfected with cDNA for RT1-E(u) but neither untransfected Rat2 nor a transfectant with the class Ia allele, RT1-A(u). Cytolysis of Rat2-RT1-E(u) was inhibited by the anti-RT1-E(u) antibody 70-3-C2. In addition, NK cells cytolytic against PVG (RT1(c)) targets, but not against WF (RT1(u)) or other allogeneic targets were activated after PVG immunization of BN rats. The generation of NK populations cytolytic for target cells of the same haplotype as the immunizing cells, but not for third-party targets, strongly suggests the existence of a selective NK-mediated response in vivo. We conclude that recognition of an allogeneic MHC class Ib RT1-E molecule activates NK cells and the specific cytolytic response could be regarded as adaptive.

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European journal of immunology, 29, 0014-2980, 3663-73, 1999

PMID: 10556822

Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor.
Nakamura MC, Naper C, Niemi EC, Spusta SC, Rolstad B, Butcher GW, Seaman WE, Ryan JC

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.

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Journal of immunology (Baltimore, Md. : 1950), 163, 0022-1767, 4694-700, 1999

PMID: 10528166

Open Access

Greatly reduced efficiency of both positive and negative selection of thymocytes in CD45 tyrosine phosphatase-deficient mice.
Mee PJ, Turner M, Basson MA, Costello PS, Zamoyska R, Tybulewicz VL

The T cell repertoire is shaped by positive and negative selection of thymocytes. TCR-mediated signals that determine these selection processes are only partly understood. The CD45 tyrosine phosphatase has been shown to be important for signal transduction through the TCR, but there has been disagreement about whether CD45 is a positive or negative regulator of TCR signaling. Using CD45-deficient mice expressing transgenic TCR, we show that in the absence of CD45 there is a large increase in the thresholds of TCR stimulation required for both positive and negative selection. Our results conclusively demonstrate that in double-positive thymocytes CD45 is a positive regulator of the TCR signals that drive thymic selection events.

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European journal of immunology, 29, 0014-2980, 2923-33, 1999

PMID: 10508267

The CD45 tyrosine phosphatase regulates CD3-induced signal transduction and T cell development in recombinase-deficient mice: restoration of pre-TCR function by active p56(lck).
Pingel S, Baker M, Turner M, Holmes N, Alexander DR

The pre-TCR complex regulates the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes during T cell development. In CD45(-/-) mice there is an accumulation of DN cells, suggesting a possible role for CD45 in pre-TCR signaling. We therefore crossed CD45(-/-) with Rag-1(-/-) mice to investigate the signaling functions of the CD3 complex in DN thymocytes. Remarkably, treatment of Rag-1(-/-)/CD45(-/-) mice with a CD3 mAb caused maturation to the DP stage at only 3% of the level measured in Rag-1(-/-) mice. Furthermore, ligation of the CD3 complex on Rag-1(-/-) /CD45(-/-) thymocytes in vitro induced less tyrosine phosphorylation in specific proteins when compared to Rag-1(-/-) thymocytes. CD45(-/-) mice were also crossed with pLGFA mice expressing a constitutively active form of the lck tyrosine kinase which restored the DN to DP transition to near normal levels. Our results are consistent with a model in which CD45-activated p56(lck) is critical for pre-TCR signal transduction.

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European journal of immunology, 29, 0014-2980, 2376-84, 1999

PMID: 10458749

Redundant role of the Syk protein tyrosine kinase in mouse NK cell differentiation.
Colucci F, Turner M, Schweighoffer E, Guy-Grand D, Di Bartolo V, Salcedo M, Tybulewicz VL, Di Santo JP

Syk and ZAP-70 subserve nonredundant functions in B and T lymphopoiesis. In the absence of Syk, B cell development is blocked, while T cell development is arrested in the absence of ZAP-70. The receptors and the signaling molecules required for differentiation of NK cells are poorly characterized. Here we investigate the role of the Syk protein tyrosine kinase in NK cell differentiation. Hemopoietic chimeras were generated by reconstituting alymphoid (B-, T-, NK-) recombinase-activating gene-2 x common cytokine receptor gamma-chain double-mutant mice with Syk-/- fetal liver cells. The phenotypically mature Syk-/- NK cells that developed in this context were fully competent in natural cytotoxicity and in calibrating functional inhibitory receptors for MHC molecules. Syk-deficient NK cells demonstrated reduced levels of Ab-dependent cellular cytotoxicity. Nevertheless, Syk-/- NK cells could signal through NK1. 1 and 2B4 activating receptors and expressed ZAP-70 protein. We conclude that the Syk protein tyrosine kinase is not essential for murine NK cell development, and that compensatory signaling pathways (including those mediated through ZAP-70) may sustain most NK cell functions in the absence of Syk.

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Journal of immunology (Baltimore, Md. : 1950), 163, 0022-1767, 1769-74, 1999

PMID: 10438908

Open Access

Genes in two major histocompatibility complex class I regions control selection, phenotype, and function of a rat Ly-49 natural killer cell subset.
Naper C, Ryan JC, Kirsch R, Butcher GW, Rolstad B, Vaage JT

We have generated a monoclonal antibody (STOK2) which reacts with an inhibitory MHC receptor on a subset of alloreactive NK cells in the rat. This receptor, termed the STOK2 antigen (Ag), belongs to the Ly-49 family of lectin-like molecules and displays specificity for the classical MHC class I molecule RT1-A1c of PVG rats. Here, we have investigated the influence of the MHC on the selection, phenotype and function of the STOK2+ NK subset in a panel of MHC congenic and intra-MHC recombinant strains. STOK2 receptor density was influenced by the presence of its classical MHC I ligand RT1-A1c, as evidenced by a reduction of STOK2 Ag on the surface of NK cells from RT1-A1c+, as compared with RT1-A1c-, strains. In addition, a role for nonclassical MHC I RT1-C/E/M alleles in the selection of the STOK2 Ag was demonstrated. The relative number of STOK2+ NK cells was fivefold higher in rats expressing the RT1-C/E/M(av1) as compared with those expressing the RT1-C/E/M(u) class Ib haplotype. The STOK2 ligand RT1-A1c inhibited cytotoxicity of STOK2+ NK cells regardless of effector cell MHC haplotype. Allospecificity of STOK2+ NK cells varied markedly with effector cell MHC, however, and suggested that inhibitory MHC I receptors apart from STOK2 were variably co-expressed by these cells. These data provide evidence for the MHC-dependent regulation of the allospecific repertoire within a subset of potentially autoreactive Ly-49+ rat NK cells.

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European journal of immunology, 29, 0014-2980, 2046-53, 1999

PMID: 10382768

The lymphopenia mutation of the BB rat causes inappropriate apoptosis of mature thymocytes.
G Hernández-Hoyos, S Joseph, NG Miller, GW Butcher

BB rats develop autoimmune diabetes mellitus at a high frequency. A key factor in the development of the disease is an autosomal recessive mutation determining peripheral T cell lymphocytopenia. Previous studies have suggested that the lymphopenia could be caused by increased cell death. Here we demonstrate that the lyp mutation dramatically reduces the in vitro lifespan of the TCRhi single-positive thymocytes and peripheral T cells, without abolishing their capacity to proliferate. The reduced lifespan is due to an increased rate of apoptosis, and is detected in single-positive thymocytes displaying characteristics of cells which have undergone positive selection. The cell death defect does not affect the in vitro lifespan of peripheral B cells. Interestingly, stimulation can rescue peripheral lyp/lyp T cells from immediate cell death. We propose that the lymphopenia mutation prevents the accumulation of a normal T cell pool, including regulatory subsets, without preventing the activation and proliferation of reactive T cells, thereby creating conditions appropriate for the development of uncontrolled autoimmune responses.

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European journal of immunology, 29, 6, 1832-41, 1999

PMID: 10382745

DOI: 10.1002/(SICI)1521-4141(199906)29:063.0.CO;2-F

Collagen mediates changes in intracellular calcium in primary mouse megakaryocytes through syk-dependent and -independent pathways.
Briddon SJ, Melford SK, Turner M, Tybulewicz V, Watson SP

We have characterized changes in [Ca2+]i in primary mouse megakaryocytes in response to fibrillar collagen and in response to cross-linking of the collagen receptor, the integrin alpha2beta1. The response to collagen was markedly different from that seen to a triple helical collagen-related peptide (CRP), which signals via the tyrosine kinases p59(fyn) and p72(syk). This peptide binds to the collagen receptor glycoprotein VI (GPVI), but not to the integrin alpha2beta1. Collagen elicited a sustained increase in [Ca2+]i composed primarily of influx of extracellular Ca2+ with some Ca2+ release from internal stores. In contrast to CRP, this response was only partially (approximately 30%) inhibited by the src-family kinase inhibitor PP1 (10 micromol/L) or by microinjection of the tandem SH2 domains of p72(syk). Collagen also caused an increase in [Ca2+]i in megakaryocytes deficient in either p59(fyn) or p72(syk), although the response was reduced by approximately 40% in both cases: Cross-linking of the alpha2 integrin increased [Ca2+]i in these cells exclusively via Ca2+ influx. This response was reduced by approximately 50% after PP1 pretreatment, but was significantly increased in fyn-deficient megakaryocytes. Collagen therefore increases [Ca2+]i in mouse megakaryocytes via multiple receptors, including GPVI, which causes Ca2+ mobilization, and alpha2beta1, which stimulates a substantial influx of extracellular Ca2+.

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Blood, 93, 0006-4971, 3847-55, 1999

PMID: 10339492

Open Access

Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets.
Law DA, Nannizzi-Alaimo L, Ministri K, Hughes PE, Forsyth J, Turner M, Shattil SJ, Ginsberg MH, Tybulewicz VL, Phillips DR

Agonists induce inside-out alphaIIbbeta3 signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in alphaIIbbeta3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of alphaIIbbeta3 by adenine diphosphate (ADP) +/- epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in alphaIIbbeta3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits alphaIIbbeta3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.

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Blood, 93, 0006-4971, 2645-52, 1999

PMID: 10194444

Open Access

The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK, and NF-kappaB pathways.
Costello PS, Walters AE, Mee PJ, Turner M, Reynolds LF, Prisco A, Sarner N, Zamoyska R, Tybulewicz VL

Vav is a GTP/GDP exchange factor (GEF) for members of the Rho-family of GTPases that is rapidly tyrosine-phosphorylated after engagement of the T cell receptor (TCR), suggesting that it may transduce signals from the receptor. T cells from mice made Vav-deficient by gene targeting (Vav-/-) fail to proliferate in response to TCR stimulation because they fail to secrete IL-2. We now show that this is due at least in part to the failure to initiate IL-2 gene transcription. Furthermore, we analyze TCR-proximal signaling pathways in Vav-/- T cells and show that despite normal activation of the Lck and ZAP-70 tyrosine kinases, the mutant cells have specific defects in TCR-induced intracellular calcium fluxes, in the activation of extracellular signal-regulated mitogen-activated protein kinases and in the activation of the NF-kappaB transcription factor. Finally, we show that the greatly reduced TCR-induced calcium flux of Vav-deficient T cells is an important cause of their proliferative defect, because restoration of the calcium flux with a calcium ionophore reverses the phenotype.

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Proceedings of the National Academy of Sciences of the United States of America, 96, 0027-8424, 3035-40, 1999

PMID: 10077632

Open Access

Defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.
Gulbranson-Judge A, Tybulewicz VL, Walters AE, Toellner KM, MacLennan IC, Turner M

Vav, a guanine nucleotide exchange factor for members of the Rho family of small GTPases, is activated through engagement of B and T lymphocyte antigen receptors. It is important for establishing the signaling threshold of the TCR, as mice lacking Vav display defective thymocyte selection. Here, conventional B cells are shown to develop normally in Vav-deficient mice but these mice have few B-1 B cells. The threshold for inducing B cell proliferation through BCR engagement in vitro is greater in Vav-deficient B cells. Nevertheless, in vivo the mutant mice have normal antibody responses to haptenated Ficoll. In contrast, Vav-/- mice show defective class switching to IgG and germinal center formation when immunized with haptenated protein. Interestingly, this defect is reversed in chimeras where normal T cells are present. Antigen-specific proliferation of T cells in the T zone was found to be similar in wild-type and Vav-/- mice but the induction of IL-4 mRNA and switch transcripts was specifically impaired. These results suggest that defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

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European journal of immunology, 29, 0014-2980, 477-87, 1999

PMID: 10064063

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets.
Gross BS, Lee JR, Clements JL, Turner M, Tybulewicz VL, Findell PR, Koretzky GA, Watson SP

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.

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The Journal of biological chemistry, 274, 0021-9258, 5963-71, 1999

PMID: 10026222

Open Access

At least two receptors of asymmetric acetylcholinesterase are present at the synaptic basal lamina of Torpedo electric organ.
OI Casanueva, P Deprez, T García-Huidobro, NC Inestrosa

Asymmetric acetylcholinesterase (AChE) is anchored to the basal lamina (BL) of cholinergic synapses via its collagenic tail, yet the complement of matrix receptors involved in its attachment remains unknown. The development of a novel overlay technique has allowed us to identify two Torpedo BL components that bind asymmetric AChE: a polypeptide of approximately 140 kDa and a doublet of 195-215 kDa. These were found to stain metachromatically with Coomassie blue R-250, were solubilized by acetic acid, and were sensitive to collagenase treatment. Upon sequence analysis, the 140 kDa polypeptide yielded a characteristic collagenous motif. Another AChE-binding BL constituent, identified by overlay, corresponded to a heparan sulfate proteoglycan. Lastly, we established that this proteoglycan, but not the collagenous proteins, interacted with at least one heparin binding domain of the collagenic tail of AChE. Our results indicate that at least two BL receptors are likely to exist for asymmetric AChE in Torpedo electric organ.

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Biochemical and biophysical research communications, 250, 2, 312-7, 1998

PMID: 9753626

DOI: 10.1006/bbrc.1998.9303

Arabinogalactan-proteins from Nicotiana alata and Pyrus communis contain glycosylphosphatidylinositol membrane anchors.
Youl JJ, Bacic A, Oxley D

Arabinogalactan-proteins (AGPs) are a class of proteoglycans found in cell secretions and plasma membranes of plants. Attention is currently focused on their structure and their potential role in growth and development. We present evidence that two members of a major class of AGPs, the classical AGPs, AGPNa1 from styles of Nicotiana alata and AGPPc1 from cell suspension cultures of Pyrus communis, undergo C-terminal processing involving glycosylphosphatidylinositol membrane anchors. The evidence is that (i) the transmembrane helix at the C terminus predicted from the cDNA encoding these proteins is not present-the C-terminal amino acid is Asn87 and Ser97 for AGPNa1 and AGPPc1, respectively; (ii) both AGP protein backbones are substituted with ethanolamine at the C-terminal amino acid; and (iii) inositol, glucosamine, and mannose are present in the native AGPs. An examination of the deduced amino acid sequences of other classical AGP protein backbones shows that glycosylphosphatidylinositol-anchors may be a common feature of this class of AGPs.

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Proceedings of the National Academy of Sciences of the United States of America, 95, 0027-8424, 7921-6, 1998

PMID: 9653116

CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav.
O'Rourke LM, Tooze R, Turner M, Sandoval DM, Carter RH, Tybulewicz VL, Fearon DT

CD19 is a coreceptor that amplifies signaling by membrane immunoglobulin (mIg) to promote responses of the B lymphocyte to T-dependent antigens. Vav is a guanine nucleotide exchange factor for the Rho, Rac, Cdc42 family of small GTPases. We found that coligating mIg and CD19 causes a synergistic increase in the tyrosine phosphorylation of CD19. Phosphorylated tyrosine-391 of CD19 binds Vav to mediate a sustained increase in intracellular Ca2+ concentration. This response correlates with activation by the CD19-Vav complex of phosphatidylinositol 4-phosphate 5-kinase for the synthesis of phosphatidylinositol 4,5-bisphosphate. Interaction of CD19 with Vav also mediates the synergistic activation of the mitogen-activated protein kinase JNK. Therefore, CD19 is a membrane adaptor protein that recruits Vav for the activation of lipid and protein kinases.

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Immunity, 8, 1074-7613, 635-45, 1998

PMID: 9620684

Open Access

Structure and distribution of N-glycans on the S7-allele stylar self-incompatibility ribonuclease of Nicotiana alata.
Oxley D, Munro SL, Craik DJ, Bacic A

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S7-allele RNase of N. alata. A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), 1H-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides. All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans. The N-glycosylation pattern of the S7-RNase is significantly different to those of the S1- and S2-RNases.

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Journal of biochemistry, 123, 0021-924X, 978-83, 1998

PMID: 9562634

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato.
Parry S, Newbigin E, Craik D, Nakamura KT, Bacic A, Oxley D

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

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Plant physiology, 116, 0032-0889, 463-9, 1998

PMID: 9489006

Fc epsilon receptor I-associated lyn-dependent phosphorylation of Fc gamma receptor IIB during negative regulation of mast cell activation.
Malbec O, Fong DC, Turner M, Tybulewicz VL, Cambier JC, Fridman WH, Daëron M

Fc gamma RIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). Fc gamma RIIB inhibit cell activation triggered by receptors that signal via immunoreceptor tyrosine-based activation motifs. This inhibition requires ITIM tyrosyl phosphorylation and is correlated with the binding of SH2 domain-containing phosphatases that may mediate the inhibitory signal. In the present work, we investigated the mechanism of Fc gamma RIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of Fc gamma RIIB requires coaggregation with Fc epsilon RI and that, once phosphorylated, Fc gamma RIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated Fc gamma RIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of Fc gamma RIIB with Fc epsilon RI does not prevent Fc epsilon RI-mediated activation of lyn and syk. Both kinases can phosphorylate Fc gamma RIIB in vitro. However, when coaggregated with Fc epsilon RI, Fc gamma RIIB was in vivo phosphorylated in syk-deficient mast cells, but not in lyn-deficient mast cells. When Fc epsilon RI are coaggregated with Fc gamma RIIB by immune complexes, Fc epsilon RI-associated lyn may thus phosphorylate Fc gamma RIIB. By this mechanism, Fc epsilon RI initiate ITIM-dependent inhibition of intracellular propagation of their own signals.

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Journal of immunology (Baltimore, Md. : 1950), 160, 0022-1767, 1647-58, 1998

PMID: 9469421

Open Access

A major portion of synaptic basal lamina acetylcholinesterase is detached by high salt- and heparin-containing buffers from rat diaphragm muscle and Torpedo electric organ.
Casanueva OI, García-Huidobro T, Campos EO, Aldunate R, Garrido J, Inestrosa NC

Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.

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The Journal of biological chemistry, 273, 0021-9258, 4258-65, 1998

PMID: 9461624

Open Access

Identification of active-site histidine residues of a self-incompatibility ribonuclease from a wild tomato.
Parry S, Newbigin E, Currie G, Bacic A, Oxley D

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T2 family and the mechanism of S-RNase action.

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Plant physiology, 115, 0032-0889, 1421-9, 1997

PMID: 9414554

Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool.
Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL

The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

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The Journal of experimental medicine, 186, 0022-1007, 2013-21, 1997

PMID: 9396770

Open Access

A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes.
Turner M, Mee PJ, Walters AE, Quinn ME, Mellor AL, Zamoyska R, Tybulewicz VL

The T cell repertoire is shaped by positive and negative selection of thymocytes that express low levels of T cell receptor (TCR) and both CD4 and CD8. TCR-mediated signals that determine these selection processes are only partly understood. Vav, a GDP-GTP exchange factor for Rho-family proteins, is tyrosine phosphorylated following TCR stimulation, suggesting that it may transduce TCR signals. We now demonstrate that mice lacking Vav are viable and display a profound defect in the positive selection of both class I- and class II-restricted T cells. In contrast, Vav is not essential for negative selection, though in its absence negative selection is much less effective. Vav may influence the efficiency of TCR-induced selection events by regulating the intracellular calcium flux of thymocytes.

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Immunity, 7, 1074-7613, 451-60, 1997

PMID: 9354466

Open Access

Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide.
Melford SK, Turner M, Briddon SJ, Tybulewicz VL, Watson SP

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.

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The Journal of biological chemistry, 272, 0021-9258, 27539-42, 1997

PMID: 9346887

Open Access