Manfred Brockhaus

Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.

TWO HUMAN TNF RECEPTORS HAVE SIMILAR EXTRACELLULAR, BUT DISTINCT INTRACELLULAR, DOMAIN SEQUENCES

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif

Junctional adhesion molecules (JAMs) are a family of immunoglobulin‐like single‐span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 Å resolution. rsJAM consists of two immunoglobulin‐like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U‐shaped dimer with highly complementary interactions between the N‐terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U‐shaped JAM dimers are oriented in cis on the cell surface and form a two‐dimensional network by trans‐interactions of their N‐terminal domains with JAM dimers from an opposite cell surface.

Human TNF mutants with selective activity on the p55 receptor

THE remarkable ability of tumour necrosis factor (TNF), especially in combination with interferon, selectively to kill or inhibit malignant cell lines is so far unmatched by any other combination of cytokines1–4. But clinical trials in cancer patients have on the whole been disappointing5–7, and it has been estimated that a TNF dose would be effective only at 5–25 times the maximum tolerated dose4. High TNF concentrations give a much more pronounced antitumour activity in mice1,8–10, in which murine TNF is about 50-fold more systemically toxic than human TNF11,12. But there is little or no species specificity in cytotoxicity of murine TNF and human TNF on human as well as on murine cell lines13,14. This dual action of TNF may be explained by the existence of two types of receptor for TNF15,16: the smaller, TNF-R55, is present on most cells and particularly on those susceptible to the cytotoxic action of TNF17; the larger, TNF-R55, is also present on many cell types15,16, especially those of myeloid origin, and is strongly expressed on stimulated T and B lymphocytes18. In mice, human TNF binds only to murine TNF-R55 (ref. 15), which can then mediate cytotoxic activity on malignant cells15–17,19. As human TNF does not bind to murine TNF-R75, the latter must be responsible for the much enhanced systemic toxicity of murine TNF. Human TNF can, however, become toxic in mice when a second pathway is activated1,11,20. There is no reciprocal situation in the human system: human and murine TNF bind almost equally well to the two human TNF receptors. Here we describe human TNF mutants that still interact with the human TNF-R55 receptor but which have largely lost their ability to bind to human TNF-R75. Activation of TNF-R55 is sufficient to trigger cytotoxic activity towards transformed cells. One representative human TNF mutant retains its antitumour activity in nude mice carrying tumours derived from human cancers. Under the appropriate conditions, such human TNF mutants are expected to induce less systemic toxicity in man, while still exerting their direct antitumour effect.

Homophilic Interaction of Junctional Adhesion Molecule

Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.

Many monoclonal antibodies with an apparent specificity for certain lung cancers are directed against a sugar sequence found in lacto-N-fucopentaose III

Abstract Monoclonal antibodies with an apparent specificity for human small-cell carcinoma, adenocarcinoma, and squamous carcinoma of the lung are produced by some hybridomas obtained from mice and rats immunized with an established line of human small cell lung cancer. Out of 85 of these antibodies produced by independently isolated hybridomas from 15 different fusions, 21 are directed against the sugar sequence which occurs in lacto- N -fucopentaose III ceramide, in several higher glycolipids and in glycoproteins. Specificity was determined by autoradiography of thin-layer chromatograms of glycolipids, by solid-phase radioimmunoassays, and by hapten inhibition studies. All 21 antibodies are of the immunoglobulin M type.

Antibodies to the Junctional Adhesion Molecule Cause Disruption of Endothelial Cells and Do Not Prevent Leukocyte Influx into the Meninges after Viral or Bacterial Infection

A hallmark of infectious meningitis is the invasion of leukocytes into the subarachnoid space. In experimental meningitis triggered by tumor necrosis factor-alpha and interleukin-1beta, the interaction of leukocytes with endothelial cells and the subsequent migration of the cells through the vessel wall can be inhibited by an antibody to the junctional adhesion molecule (JAM). In contrast to the cytokine-induced meningitis model, anti-JAM antibodies failed to prevent leukocyte influx into the central nervous system after infection of mice with Listeria monocytogenes or lymphocytic choriomeningitis virus. Furthermore, in bacterial meningitis, anti-JAM IgG antibodies, but not Fab fragments, caused disruption of the endothelium. Likewise complement-dependent antibody-mediated cytotoxicity was observed in cultured brain endothelial cells treated with anti-JAM IgG but not with its Fab fragment.

Caspase-mediated cleavage is not required for the activity of presenilins in amyloidogenesis and NOTCH signaling

THE Alzheimers disease (AD) associated presenilin (PS) proteins are proteolytically processed. One of the processing pathways involves cleavage by caspases. Pharmacological inhibition of caspases is currently being discussed as a treatment for a variety of neurodegenerative diseases, including AD. We therefore inhibited caspase mediated processing of PS-1 and PS-2 in cells transfected with wt and mutant PS by mutagenizing the substrate recognition site or by using specific peptide aldehydes known to block caspases. We found that the inhibition of caspase mediated processing of PS proteins does not decrease its amyloidogenic activity. PS cDNA constructs with mutations in the caspase cleavage site are biologically active in Caenorhabditis elegans such as the wt human PS proteins, demonstrating that caspase-mediated cleavage is not required for the physiological PS function in NOTCH signaling.

Distribution and abundance of metabotropic glutamate receptor subtype 2 in rat brain revealed by [3H]LY354740 binding in vitro and quantitative radioautography: Correlation with the sites of synthesis, expression, and agonist stimulation of [35S]GTPγs binding

Until recently, there was a lack of selective radioligands for the subtypes of metabotropic glutamate (mGlu) receptors. [3H]LY354740 ((+)‐2‐aminobicyclo[3,1,0]hexane‐2,6‐dicarboxylic acid), a selective agonist for group II receptors (mGlu2 and ‐3, which are negatively coupled to cAMP production), has now been used to map their brain distribution and abundance by in vitro binding and quantitative radioautography. The selective cation dependence of its binding allowed the discrimination between mGlu2 and mGlu3 receptor labeling. Thus, in the presence of Ca2+ and Mg2+ ions, the agonist bound selectively to mGlu2 receptors as evidenced by: 1) the correlative distribution and abundance of binding sites (highest in the lacunosum moleculare of the hippocampus and lowest in white matter) with mGlu2 receptor mRNA and protein revealed by in situ hybridization histochemistry and immunohistochemistry, respectively; 2) its selective pharmacology; and 3) the distribution of LY354740‐stimulated [35S]GTPγS binding (25–97% above basal, according to the brain region), revealing G protein‐coupled receptor coupling to Gi proteins. Nonspecific binding (in the presence of 10 μM DCG‐IV, a group II‐selective, mGlu2‐preferring, receptor agonist) was <10% of total. In adjacent sections, the distribution of binding sites for [3H]DCG‐IV was very similar. This extensive study paves the way for investigations of the regional expression and regulation of mGlu2 receptors in human CNS diseases, such as Alzheimers disease, which may reveal their functional roles and identify potential therapeutic drug targets. Indeed, it has recently been demonstrated (Higgins et al. [2004] Neuropharmacology 46:907–917) that pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent. J. Comp. Neurol. 487:15–27, 2005.

Evaluation of soluble tumor necrosis factor (TNF) receptors and TNF receptor antibodies in patients with systemic lupus erythematodes, progressive systemic sclerosis, and mixed connective tissue disease

Two TNF binding proteins have been characterized as soluble fragments of TNF receptors. We measured the plasma concentrations of soluble type A (p75) and type B (p55) TNF receptors in patients with systemic lupus erythematodes (SLE), progressive systemic sclerosis (PSS), and mixed connective tissue disease (MCTD). In SLE and PSS patients plasma concentrations of both types of TNF receptors and in MCTD patients type A TNF receptors were significantly elevated compared to controls. Plasma concentrations of both soluble TNF receptors were highly correlated in SLE, PSS, and MCTD patients, indicating a possible coregulation of both TNF receptors. In contrast, soluble interleukin 2 receptor (sCD 25) plasma concentrations were not correlated and seem to be an independent parameter. The soluble forms of the TNF receptors neutralize TNF in cytotoxicity assays and are functionally active as TNF antagonists. In one patient with SLE, autoantibodies against type A TNF receptors were detected, TNFα, and TNFβ did not interfere with the autoantibody binding to the receptor.