Probing ligand-induced conformational changes of human CD38.

The lymphoid surface antigen CD38 is basically a NAD+glycohydrolase, which is also involved in the metabolism of cyclic ADP-ribose. Besides, this ecto-enzyme has potential signalling roles in T- and B-cells. Such multiple functions prompted us to study the molecular dynamics of the CD38 protein and especially the relationship between its ecto-enzymatic active site and its epitope, i.e. the binding site of most known anti-CD38 monoclonal antibodies. Both epitopic and enzymatic sites were shown to be degraded by proteases, such as trypsin or chymotrypsin. This sensitivity was almost entirely suppressed in the presence of substrates or inhibitors. Both sites were also degraded in the presence of reducing agents, as dithiothreitol. Inhibitory ligands induced the same resistance of both sites against reducing attack. The binding of CD38 ligands to the active site triggers therefore conformational changes that shield some backbone bonds and disulfide bridges against, respectively, proteolytic cleavage or reduction. This transconformation was found moreover to irreversibly take place after incubation with substrates such as NAD+ in the presence of dithiothreitol. The epitope remained preserved, while the enzymatic activity was lost. This inactivation probably resulted from the covalent trapping of the catalytically reactive intermediate in the active site (i.e. paracatalytic inactivation). These data have major implications in the knowledge of the CD38 structure, especially with regard to the location of disulfide bridges and their accessibility. Potential consequences of the conformational plasticity of CD38 should also be considered in its physiological functions such as signalling.

Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry.

BACKGROUND: The kinetics of protein-protein interactions can be monitored with optical biosensors based on the principles of either surface plasmon resonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane. METHODS: We monitored with an IASys biosensor system, based on a resonant mirror: (1) the binding of cells to an immobilized ligand, (2) the binding of a soluble ligand to immobilized cells, and (3) the binding of a soluble ligand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic flow cytometry. RESULTS: With an optical biosensor, the useful configuration is the one based on immobilized plasma membrane vesicles. However, signals can be detected only for very abundant binding sites (>10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor. CONCLUSIONS: Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode.

Upregulation of CD38 gene expression in leukemic B cells by interferon types I and II.

The activation antigen CD38, which has NAD+ glycohydrolase activity in its extracellular domain, is expressed by a large variety of cell types. Few investigations into the regulation of CD38 expression by physiologic stimuli have been reported. As the CD38 promoter contains potential binding sites for interferon (IFN) regulatory factor-1 (IRF-1), we investigated the influence of IFN type I (alpha and beta) and type II (gamma) on CD38 gene expression of leukemic B cells. Using the IFN-responsive B cell line Eskol, we found by RT-PCR analysis a rapid time-dependent induction in CD38 mRNA (starting at 6 h) with each type of IFN. This induction was independent of protein synthesis, suggesting that CD38 gene activation does not require IRF-1 but is merely under direct transcriptional regulation by latent IFN-inducible factors. mRNA stimulation was followed within 24 h by induction of membrane CD38, which coincided with rises of CD38-specific ectoenzymatic activities, that is, NAD+ glycohydrolase, (A/G)DP-ribosyl cyclase, and cyclic ADP ribose hydrolase activities. IFN failed to induce or upregulate the other CD38-related ectoenzymes analyzed, that is, CD39, CD73, CD157, and PC-1. Similarly, treatment of leukemic cells of patients with B chronic lymphocytic leukemia (B-CLL) with IFN resulted in an increase in CD38 mRNA mirrored by plasma membrane upregulation of CD38 and NAD+ glycohydrolase activity. Further investigation in relation to CD38 gene activation and B-CLL behavior remains to be defined.

[Stimulation of C-fos and jun B proto-oncogenes: potential role of TRH effects in clone cell line with prolactin (GH3B6)]. / La stimulation des proto-oncogènes c-fos et jun B: un relais potential des effets du TRH dans une lignée clonale de cellules à prolactine (GH3B6).

The hypothalamic neuropeptide TRH, which stimulates prolactin (PRL) release and PRL gene transcription, also raises c-fos proto-oncogene mRNA levels in GH3B6 rat pituitary cells. C-fos is assumed to be involved in the transduction of external signals to the nucleus as a component of AP1 transcription factor, a protein complex that contains a member of the jun proto-oncogene family. We have thus looked for the member(s) of the jun family that could be the partner of c-fos in TRH-stimulated GH3B6 cells. The common biphasic pattern of jun B and c-fos mRNA regulation under TRH exposure, i.e., an early peak and a long-lasting plateau phase, suggested that jun B was the best candidate. Then, to better understand the mode of action of TRH and to look for possible functions of c-fos and jun B in these cells, we have investigated the role of different intracellular signalings in the induction of each proto-oncogene. This was done taking as a model that the effects of TRH on PRL release and PRL gene transcription has been previously ascribed to the coupling of the TRH receptor to the activation of both protein kinase C- and calcium-dependent mechanisms. An extensive pharmacological analyses revealed that PKC-, Ca2+ but also protein kinase A-dependent mechanisms are involved in TRH-induced c-fos and jun B mRNA early responses in GH3B6 cells. The overall study also revealed specific features in the control by TRH of each proto-oncogene by some intracellular messengers. Finally, considering the fact that second long lasting phase of proto-oncogene expression was found associated with increased PRL mRNA accumulation whatever the stimulus, it might be proposed that AP1 [c-Fos/Jun B] factor could be involved in the regulation of PRL gene expression. Such hypothesis was furthermore supported by preliminary gel-shift experiments. Nevertheless, in view of the systematic coincidence between acute PRL release and early proto-oncogene induction, a role for c-fos and jun B in the control of genes involved in the secretory process might also be suggested.

Multiple intracellular signallings are involved in thyrotropin-releasing hormone (TRH)-induced c-fos and jun B mRNA levels in clonal prolactin cells.

In mammosomatotropes GH3B6 cells, one of the primary responses to thyrotropin-releasing hormone (TRH) is the parallel induction of two proto-oncogenes, c-fos and jun B, which code for constituents of AP1 transcription factor. To better understand the mode of action of TRH and to look for possible functions of c-fos and jun B in these cells, we have investigated the role of different intracellular signals in the induction of each proto-oncogene on the one hand, and on prolactin (PRL) release and PRL gene expression on the other hand. Northern and dot-blot analyses revealed that the activation of protein kinase C (PKC)-, Ca(2+)- or adenylyl cyclase-dependent pathways acutely increased both c-fos and jun B transcripts. However, a gene specific responsiveness was revealed using phorbol 12-myristate 13-acetate (TPA) and several combined treatments. The simultaneous activation of PKC and Ca(2+)-dependent pathways resulted in synergistic stimulations of c-fos mRNA levels only. Consistently, ionomycin plus low doses of TPA solely reproduced the potent effect of TRH on c-fos transcripts. Data collected from TRH and TPA down-regulated cells indicated that TRH probably recruits TPA-dependent PKC isoforms for stimulating c-fos but not jun B transcripts. On the contrary, the TRH-induced stimulation of either proto-oncogene likely involves Ca(2+)-dependent mechanisms because calcium agonists and the peptide exert non-additive effects. Finally, the synergistic stimulations observed in response to TRH combined with forskolin, indicate that adenylyl cyclase-dependent mechanisms are interconnected with TRH-induced proto-oncogene expression. The overall study also reveals that among the agonists tested, the dihydropyridine Bay K 8644 and forskolin only were capable to induce a long-lasting stimulation of c-fos and jun B mRNA levels, concomitant to increased levels of PRL transcripts, as does TRH. Considering that AP1 is assumed to be involved in signal transmission from the cell surface to the nucleus, it might be thus proposed that a common stimulation of c-fos and jun B gene expression is possibly involved in the activation of the PRL gene. On the other hand, the systematic coincidence between acute PRL release and proto-oncogenes expression suggest a role for c-fos and jun B in the control of genes involved in the secretory process.

Distributions of molecular forms of acetylcholinesterase and butyrylcholinesterase in nervous tissue of the cat.

We analyzed the activities of acetylcholinesterase and butyrylcholinesterase, and of the metabolic enzymes enolase and lactate dehydrogenase, in the superior cervical ganglion, ciliary ganglion, dorsal root ganglion, stellate ganglion, and caudate nucleus of the cat; we found that these tissues possess very different levels of enzymic activities. The proportions of the alpha alpha, alpha gamma, and gamma gamma enolase isozymes are also quite variable. We particularly studied the molecular forms of acetylcholinesterase and butyrylcholinesterase, in normal tissues and in preganglionically denervated SCG, in comparison with earlier histochemical findings. The results are consistent with the premise that the G1 (globular monomer) forms of both enzymes are located in the cytoplasm, the G4 (globular tetramer) forms are at the plasma membranes, and the A12 (collagen-tailed, asymmetric dodecamer) form of acetylcholinesterase is at synaptic sites.

Monoclonal antibodies against acetylcholinesterase from electric organs of Electrophorus and Torpedo.

We studied the reactivity of monoclonal antibodies (mAbs) raised against acetylcholinesterase (AChE) purified from Electrophorus and Torpedo electric organs. We obtained IgG antibodies (Elec-21, Elec-106, Tor-3E5, Tor-ME8, Tor-1A5), all of them directed against the catalytic subunit of the corresponding species, with no significant cross-reactivity. These antibodies do not inhibit the enzyme and recognize all molecular forms, globular (G) and asymmetric (A). Tor-ME8 reacts specifically with the denatured A and G subunits of Torpedo AChE, in immunoblots. Several hybridomas raised against Electrophorus AChE produced IgM antibodies (Elec-39, Elec-118, Elec-121). These antibodies react with the A forms of Electrophorus electric organs and also with a subset of dimers (G2) from Torpedo electric organ. In addition, they react with a number of non-AChE components, in immunoblots. In contrast, they do not recognize AChE from other Electrophorus tissues or A forms from Torpedo electric organs.