Biomedical subjects

E L Reinherz

Publications and source records attributed to E L Reinherz.

At least 19 recordsLinked to original sources

PubMed2001

Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction.

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.

Amino Acid Sequence↗

PubMed2001

A critical role for CD2 in both thymic selection events and mature T cell function.

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2(-/-) H-2(b) mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2K(b) molecule were compared on a wild-type or CD2(-/-) background. In N15tg RAG-2(-/-) CD2(-/-) mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4(-)CD8(-) double-negative thymocytes at the CD25(+)CD44(-) stage. Moreover, mature N15tg RAG-2(-/-) CD2(-/-) T cells are approximately 100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-gamma production. Repertoire analysis shows substantial differences in Valpha usage between non-tg C57BL/6 (B6) and B6 CD2(-/-) mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.

Animals↗

PubMed2000

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160.

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.

Animals↗

PubMed2000

Heterodimeric CD3epsilongamma extracellular domain fragments: production, purification and structural analysis.

The CD3 polypeptides (epsilon, gamma, and delta) are non-covalently associated signaling subunits of the T cell receptor which form non-disulfide linked epsilongamma and epsilondelta heterodimers. With the goal of investigating their structure, Escherichia coli expression was utilized to produce CD3 ectodomain fragments including the murine CD3epsilon subunit N-terminal Ig-like extracellular domain alone or as a single chain construct with that of CD3gamma. The latter links the CD3gamma segment to the C terminus of the CD3epsilon segment via a 26 amino acid peptide (scCD3epsilongamma26). Although CD3epsilon could be produced at high yield when directed to inclusion bodies, the refolded monomeric CD3epsilon was not native as judged by monoclonal antibody binding using surface plasmon resonance and was largely unstructured by (15)N-(1)H two-dimensional NMR analysis. In contrast, scCD3epsilongamma26 could be refolded readily into a native state as shown by CD, NMR and mAb reactivity. The linker length between CD3epsilon and CD3gamma is critical since scCD3epsilongamma16 containing a 16 residue connector failed to generate a stable heterodimer. Collectively, the results demonstrate that: (i) soluble heterodimeric fragments of CD3 can be produced; (ii) cotranslation of CD3 chains insures proper folding even in the absence of the conserved ectodomain stalk region (CxxCxE); and (iii) CD3epsilon has a more stable tertiary protein fold than CD3gamma.

Amino Acid Motifs↗

PubMed2000

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes.

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Thymic selection is influenced by subtle structural variation involving the p4 residue of an MHC class I-bound peptide.

The T lineage repertoire is shaped by opposing processes of positive and negative selection. To probe the specificity of selection, N15 TCR-transgenic (tg) recombinase-activating gene (RAG)-2(- / -) H-2(b) mice recognizing the VSV8 octapeptide RGYVYQGL bound to K(b) were utilized in conjunction with VSV8 variants differing only at the central p4 position. The V4I mutant octamer, like VSV8, induces negative selection of immature double-positive thymocytes on the beta(2)-microglobulin (beta(2)M)(+ / +) background and is a strong agonist for mature N15 T cells. In contrast, V4L or V4norvaline octamers promote positive selection in N15tg RAG-2(-/-) beta(2)M(-/-) H-2(b) fetal thymic organ culture and are weak agonists for N15 T cells. Hence, the absence of a p4 side chain Cbeta-methyl group results in positive selection of the N15 TCR. Hydrophobicity of the p4 residues also modulates thymocyte fate: the positively selecting norvaline and leucine variants have one and two Cgamma-methyl groups, respectively, while the weakly selecting gamma-methylleucine p4 contains three Cgamma-methyl groups. Moreover, the most hydrophobic octamer containing p4 cyclohexylglycine substitution fails to select. Thus, for N15 and presumably other MHC class I-restricted TCR, there is a high degree of structural specificity to peptide-dependent thymic selection processes.

Animals↗

PubMed2000

Human CD4 residue Phe 43 is critical for repertoire development and maturation of MHC class II restricted CD4 single-positive T lineage cells in vivo.

To determine the functional significance of structural alteration of CD4-MHC class II interaction in vivo, two human (h)CD4-transgenic (tg) mice were established on a murine (m)CD4(-/-) H-2(b) background. The MHC class II binding-competent hCD4 (R240AhCD4) rescues the number and helper activity of hCD4(+)CD8(-) single-positive (SP) mature T cells in mCD4(-/-) mice. In contrast, the MHC class II binding-deficient F43I hCD4 mutant cannot facilitate normal differentiation of double-positive thymocytes to CD4(+)CD8(-) SP thymocytes. Hence, only 20 - 25% of CD4(+)CD8(-) SP T cells found in wild-type or R240A hCD4tg mice are generated, with resultant diminished helper responses. Differentiation of F43I hCD4 SP T cells is MHC class II but not class I dependent as demonstrated by crossing F43I hCD4tg mice onto MHC-deficient mice. These cells show a different pattern of TCR Valpha and Vbeta gene usage relative to comparable R240A hCD4 SP T cells from R240 AhCD4tg animals. Expression of activation markers including CD25 and CD69 on F43I hCD4 SP T cells suggests that autoreactive specificites may not have been eliminated intrathymically. Collectively, the results show that CD4-MHC class II interaction significantly influences intrathymic repertoire selection.

Animals↗

PubMed2000

A critical role for p59(fyn) in CD2-based signal transduction.

TCR- but not CD2-triggered IL-2 production is p56(lck) dependent. To test the hypothesis that p59(fyn), a second src-family protein tyrosine kinase (PTK) expressed in T lymphocytes, might be an essential upstream component of the CD2 signaling pathway, we generated human (h) CD2 transgenic (tg) fyn(+/+) and fyn(-/-) mice. Clustering of hCD2 molecules on resting peripheral T lymphocytes results in Ca(2+) mobilization, activation of MAPK and cellular proliferation. In contrast, in the absence of p59(fyn), these CD2-initiated activities are markedly reduced, while TCR-triggered proliferation is unaffected. Several CD2 pathway components regulated by p59(fyn) have been identified including phospholipase C-gamma1 (PLC-gamma1), Vav, protein kinase C-theta isoform (PKC-theta), docking protein (Dok), focal adhesion kinase (FAK) and Pyk2. Decreased inducible PKC-theta catalytic activity and Vav phosphorylation likely account for diminished p38 and JNK activation in hCD2tg fyn(-/-) mice. Moreover, deficiency in fyn-dependent PLC-gamma1 catalytic activity may contribute to reduced PKC-alpha-dependent ERK activation. Of note, CD2-dependent Dok but not linker from activated T cells (LAT) tyrosine phosphorylation requires p59(fyn). Furthermore, that FAK and Pyk2 are target substrates implies that p59(fyn) may be an important regulator of T cell adhesion as well. Collectively, these data identify p59(fyn) as a key PTK in CD2-mediated activation of mature T lymphocytes.

Animals↗

PubMed2000

Two YxxL segments of a single immunoreceptor tyrosine-based activation motif in the CD3zeta molecule differentially activate calcium mobilization and mitogen-activated protein kinase family pathways.

Immunoreceptor tyrosine-based activation motifs (ITAM), consisting of two YxxL segments, transmit signals leading to IL-2 gene activation in T cells. We investigated here the functional difference in signal transduction between these two YxxL segments in the CD3zeta membrane-proximal ITAM. N-terminal YxxL mutants failed to induce ZAP-70 phosphorylation, elevation of intracellular Ca2+ concentration ([Ca2+]i) or extracellular signal-regulated kinase (ERK) activation even in the presence of CD28 co-stimulation, whereas a mutant of the leucine residue at the C-terminal YxxL segment retained the ability to induce these events although this mutation abrogated the ability to induce IL-2 gene activation. In marked contrast to ERK activation, c-Jun N-terminal kinase (JNK) activation was observed in all mutants when co-stimulated with CD28. The mutant of the leucine residue at the C-terminal YxxL segment had a defect in the transcriptional activation at the NF-AT cis-element, which was restored to wild-type level by addition of a Ca2+ ionophore, suggesting that the intensity and/or duration of [Ca2+]i elevation defines the threshold of T cell activation in this mutant. Our data collectively indicate that the activation pathways of ERK, JNK and Ca2+ mobilization are differentially regulated through YxxL segments of an ITAM.

Amino Acid Sequence↗

PubMed2000

Structural basis of cell-cell interactions in the immune system.

During the past year, advances in our understanding of receptor-ligand interactions between opposing cell surfaces have occurred at a structural level. These include adhesion involving CD2-CD58, antigen-specific T-cell receptor interactions with peptides bound to major histocompatibility complex molecules (both pMHCI and pMHCII), the CD8alphaalpha co-receptor-pMHCI interaction and the binding of two distinct classes of natural killer receptors to self-MHC ligands.

Antigens, CD↗

PubMed1999

The crystal structure of a T cell receptor in complex with peptide and MHC class II.

The crystal structure of a complex involving the D10 T cell receptor (TCR), 16-residue foreign peptide antigen, and the I-Ak self major histocompatibility complex (MHC) class II molecule is reported at 3.2 angstrom resolution. The D10 TCR is oriented in an orthogonal mode relative to its peptide-MHC (pMHC) ligand, necessitated by the amino-terminal extension of peptide residues projecting from the MHC class II antigen-binding groove as part of a mini beta sheet. Consequently, the disposition of D10 complementarity-determining region loops is altered relative to that of most pMHCI-specific TCRs; the latter TCRs assume a diagonal orientation, although with substantial variability. Peptide recognition, which involves P-1 to P8 residues, is dominated by the Valpha domain, which also binds to the class II MHC beta1 helix. That docking is limited to one segment of MHC-bound peptide offers an explanation for epitope recognition and altered peptide ligand effects, suggests a structural basis for alloreactivity, and illustrates how bacterial superantigens can span the TCR-pMHCII surface.

Animals↗

PubMed1999

Expression, purification, and functional analysis of murine ectodomain fragments of CD8alphaalpha and CD8alphabeta dimers.

Soluble mouse CD8alphaalpha and CD8alphabeta dimers corresponding to the paired ectodomains (CD8(f)) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8alphaalpha(f) for H-2K(b) as measured by BIAcore revealed a approximately 65 microM K(d), similar to that of CD8alphabeta(f). Consistent with this result, CD8alphaalpha(f) as well as CD8alphabeta(f) blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8alphabeta versus CD8alphaalpha co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8alphaalpha, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.

Animals↗

PubMed1999

Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors.

Interaction between CD2 and its counterreceptor, CD58 (LFA-3), on opposing cells optimizes immune recognition, facilitating contacts between helper T lymphocytes and antigen-presenting cells as well as between cytolytic effectors and target cells. Here, we report the crystal structure of the heterophilic adhesion complex between the amino-terminal domains of human CD2 and CD58. A strikingly asymmetric, orthogonal, face-to-face interaction involving the major beta sheets of the respective immunoglobulin-like domains with poor shape complementarity is revealed. In the virtual absence of hydrophobic forces, interdigitating charged amino acid side chains form hydrogen bonds and salt links at the interface (approximately 1200 A2), imparting a high degree of specificity albeit with low affinity (K(D) of approximately microM). These features explain CD2-CD58 dynamic binding, offering insights into interactions of related immunoglobulin superfamily receptors.

Amino Acid Sequence↗

PubMed1999

Functional glycan-free adhesion domain of human cell surface receptor CD58: design, production and NMR studies.

A general strategy is presented here for producing glycan-free forms of glycoproteins without loss of function by employing apolar-to-polar mutations of surface residues in functionally irrelevant epitopes. The success of this structure-based approach was demonstrated through the expression in Escherichia coli of a soluble 11 kDa adhesion domain extracted from the heavily glycosylated 55 kDa human CD58 ectodomain. The solution structure was subsequently determined and binding to its counter-receptor CD2 studied by NMR. This mutant adhesion domain is functional as determined by several experimental methods, and the size of its binding site has been probed by chemical shift perturbations in NMR titration experiments. The new structural information supports a 'hand-shake' model of CD2-CD58 interaction involving the GFCC'C" faces of both CD2 and CD58 adhesion domains. The region responsible for binding specificity is most likely localized on the C, C' and C" strands and the C-C' and C'-C" loops on CD58.

Amino Acid Sequence↗

PubMed1999

The GYF domain is a novel structural fold that is involved in lymphoid signaling through proline-rich sequences.

T cell activation through the CD2 cell surface receptor is transmitted by proline-rich sequences within its cytoplasmic tail. A membrane-proximal proline-rich tandem repeat, involved in cytokine production, is recognized by the intracellular CD2 binding protein CD2BP2. We solved the solution structure of the CD2 binding domain of CD2BP2, which we name the glycine-tyrosine-phenylalanine (GYF) domain. The GYF sequence is part of a structurally unique bulge-helix-bulge motif that constitutes the major binding site for the CD2 tail. A hydrophobic surface patch is created by motif residues that are highly conserved among a variety of proteins from diverse eukaryotic species. Thus, the architecture of the GYF domain may be widely used in protein-protein associations.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor.

Using NMR spectroscopy, we determined the solution structure of a single-chain T-cell receptor (scTCR) derived from the major histocompatibility complex (MHC) class II-restricted D10 TCR. The conformations of complementarity-determining regions (CDRs) 3beta and 1alpha and surface properties of 2alpha are different from those of related class I-restricted TCRs. We infer a conserved orientation for TCR V(alpha) domains in complexes with both class I and II MHC-peptide ligands, which implies that small structural variations in V(alpha) confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond time scale) provides insight into immune recognition and selection mechanisms.

Amino Acid Sequence↗

PubMed1998

A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion.

A human CD2 cytoplasmic tail-binding protein, termed CD2BP1, was identified by an interaction trap cloning method. Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing. Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C-terminal SH3 domain. Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300-309), its association is augmented markedly by the CD2BP1 N-terminal segment. Upon ligand-induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co-localizing with CD2. In turn, CD2-stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)-PEST to CD2. These findings offer the first molecular view into the control processes for T cell adhesion.

Alternative Splicing↗

PubMed1998

Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation.

An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also found in several yeast and Caenorhabditis elegans proteins of unknown function. In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains regulates protein-protein interactions.

Adaptor Proteins, Signal Transducing↗