Basal and antigen-induced exposure of the proline-rich sequence in CD3ε

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.     

Evidence of positive selection on the Atlantic salmon CD3γδ gene

Atlantic salmon are typically anadromous, spending the majority of their lifetime in oceans and returning to fresh water to breed. This diversity of environments likely results in strong selective forces shaping their genome. In this paper, we present the first genomics approach to detect positive selection operating on the Salmo salar (salmon) lineage, an important aquaculture species. We identify a panel of candidate genes that may have been subject to adaptive evolution in this species. In particular, we identify a robust signature of positive selection operating on the salmon CD3γδ gene, which encodes one of the protein chains essential for formation of the T-cell receptor complex and for T-cell activation. Furthermore, we identified the particular codon sites that have been subject to positive selection in fish and highlight two sites flanking an important N-glycosylation site in this molecule. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Identification of CD3ε, CD4, CD8β splice variants of Atlantic salmon

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.     

Soluble CD36- a marker of the (pathophysiological) role of CD36 in the metabolic syndrome?

CD36 is a class B scavenger receptor observed in many cell types and tissues throughout the body. Recent literature has implicated CD36 in the pathogenesis of metabolic dysregulation such as found in obesity, insulin resistance, and atherosclerosis. Genetic variation at the CD36 loci have been associated with obesity and lipid components of the metabolic syndrome, with risk of heart disease and type 2 diabetes. Recently, non-cell bound CD36 was identified in human plasma and was termed soluble CD36 (sCD36). In this review we will describe the functions of CD36 in tissues and address the role of sCD36 in the context of the metabolic syndrome. We will also highlight recent findings from human genetic studies looking at the CD36 locus in relation to metabolic profile in the general population. Finally, we present a model in which insulin resistance, oxLDL, low-grade inflammation and liver steatosis may contribute to elevated levels of sCD36.     

Profiling of the tetraspanin CD151 web and conspiracy of CD151/integrin β1 complex in the progression of hepatocellular carcinoma

Tetraspanin CD151 has been implicated in metastasis through forming complexes with different molecular partners. In this study, we mapped tetraspanin web proteins centered on CD151, in order to explore the role of CD151 complexes in the progression of hepatocellular carcinoma (HCC). Immunoprecipitation was used to isolate tetraspanin complexes from HCCLM3 cells using a CD151 antibody, and associated proteins were identified by mass spectrometry. The interaction of CD151 and its molecular partners, and their roles in invasiveness and metastasis of HCC cells were assayed through disruption of the CD151 network. Finally, the clinical implication of CD151 complexes in HCC patients was also examined. In this study, we identified 58 proteins, characterized the tetraspanin CD151 web, and chose integrin β1 as a main partner to further investigate. When the CD151/integrin β1 complex in HCC cells was disrupted, migration, invasiveness, secretion of matrix metalloproteinase 9, and metastasis were markedly influenced. However, both CD151 and integrin β1 expression were untouched. HCC patients with high expression of CD151/integrin β1 complex had the poorest prognosis of the whole cohort of patients. Together, our data show that CD151 acts as an important player in the progression of HCC in an integrin β1-dependent manner.     

Basis of the intrinsic flexibility of the Cε3 domain of IgE

Allergic reactions are triggered by the interaction between IgE and its high-affinity receptor, FcεRI. Various studies have mapped the interaction surface between IgE and its cellular receptors to the third constant domain of IgE (Cε3). The isolated Cε3 domain has been shown to exist as a molten globule, and the domain retains significant flexibility within the context of the IgE protein. Here we have analyzed the structural basis of the intrinsic flexibility of this domain. We have compared the sequence of the Cε3 domain to the sequences of other members of the C1 subset of the immunoglobulin superfamily and observed that Cε3 has an unusually high electrostatic charge and an unusually low content of hydrophobic residues. Mutations restoring Cε3 to a more canonical sequence were introduced in an attempt to derive a more structured domain, and several mutants display decreased levels of disorder. Engineered domains of Cε3 with a range of structural rigidities could serve as important tools for the elucidation of the role of flexibility of the Cε3 domain in IgE's biological functions.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73⁺ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses.     

Crosslinking of Fas/CD95 suppresses the CD3-mediated signaling events in Jurkat T cells by inhibiting the association of the T-cell receptor ζ chain with src-protein tyrosine kinases and ZAP70

Crosslinking of Fas (APO-1/CD95) on the surface of T cells initiates a biochemical cascade leading to programmed cell death. We have previously shown that crosslinking of Fas with an apoptosis-inducing IgM anti-Fas mAb results in suppression of the CD3-initiated cell signaling including Ca²⁺ mobilization and protein tyrosine phosphorylation. We conducted experiments to decipher the mechanisms whereby the cross talk between the Fas- and CD3 signaling pathways occur. We used lysates from Jurkat T and examined the composition of the TCR chain-precipitated immune complexes using immunoblots. While crosslinking of Fas affected the association of p59^fyn and p56^lck tyrosine kinases with the TCR chain to a limited degree, it dramatically inhibited the association of the protein tyrosine kinase ZAP70 with the chain. In cells that were preincubated with an apoptosis-inducing anti-Fas mAb, the binding of the protein tyrosine phosphatases SHP-1 to the TCR chain was increased. These experiments indicate that crosslinking of Fas interferes with early T cell signaling events by promoting the recruitment of SHP-1 and decreasing the association of protein tyrosine kinases with TCR chain. Therefore, crosslinking of Fas antigen may regulate the antigen-induced T cell response and play an active role in the T cell anergy.     

Catalytic roles of the AMP at the 3′ end of tRNAs

Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3 terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3 terminus of tRNA.     

The Gene Expression Profile of CD11c+CD8α? Dendritic Cells in the Pre-Diabetic Pancreas of the NOD Mouse

Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c⁺CD8α⁻ DCs (strong CD4+ T cell proliferation inducers) and the CD8α⁺CD103⁺ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c⁺CD8α⁻ DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c⁺CD8α⁻ DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c⁺CD8α⁻ DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c⁺CD8α⁻ DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c⁺CD8α⁻ DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.