Christophe Bignon

Crystal Structure of Thermotoga Maritima Alpha-L-Fucosidase. Insights Into the Catalytic Mechanism and the Molecular Basis for Fucosidosis.

Fucosylated glycoconjugates are involved in numerous biological events, and α-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in α-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into α-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima α-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)8-like domain and a C-terminal β-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp244, and the Brønsted acid/base, Glu266. Because T. maritima α-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.

Prolactin signal transduction to milk protein genes: carboxy-terminal part of the prolactin receptor and its tyrosine phosphorylation are not obligatory for JAK2 and STAT5 activation.

In this study, we have developed several Chinese Hamster ovary (CHO) cell clones stably expressing various deletion mutant forms of the rabbit prolactin receptor (rbPRL-R) to better define the domains of the receptor involved in JAK2 kinase interaction, STAT5 activation, and to assess the role of tyrosine phosphorylation of the PRL-R in signal transduction. We observed that the box 1 region of the receptor was critical for productive interaction with JAK2 and its tyrosine phosphorylation after PRL stimulation. However, this region appeared to require the presence of additional cytoplasmic domain region(s), such as box 2, to exert its complete effect. In addition, we found that a mutant form lacking the 141 C-terminal residues lost the capacity to be tyrosine phosphorylated in response to PRL but remained able to activate JAK2 kinase and STAT5 transcription factor, indicating that it contained the minimal sequence required for STAT5 activation. The absence of tyrosine phosphorylation of this C-terminal rbPRL-R mutant upon PRL stimulation indicated that the phosphorylation of the PRL-R normally occured in the last 141 animo acids (aa) containing three tyrosines and was not absolutely necessary for induction of these early events in PRL signal transduction. Transfectant cell lines expressing wild type (WT) PRL-R and this C-terminal mutant form were able to induce CAT activity upon PRL stimulation when transiently transfected with the ovine-beta-lactoglobulin promoter, containing STAT5 recognition sites, fused to the CAT reporter gene. The comparison between transcriptional activity of these two receptor forms leads to the conclusion that the C-terminal region of the rbPRL-R, containing the physiological sites for tyrosine phosphorylation, is probably responsible for an amplification of the PRL signal to milk protein genes.

Preparation and characterization of recombinant prolactin receptor extracellular domain from rat

Complementary (c)DNA of the extracellular domain of rat prolactin receptor (rPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A, and expressed in Escherichia coli following induction with isopropyl-b-D-thiogalactopyranoside. The expressed rPRLR-ECD protein, contained within the refractile body pellet was solubilized in 4.5 M urea, refolded and purified on a Q-Sepharose column by stepwise elution with NaCl. Only approximately 10% of the expressed protein refolded as a monomeric fraction, yielding 5-6 mg/l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Its molecular mass, determined by SDS-PAGE in the absence of reducing agent, was 28 kDa, and by gel filtration, 25.6 kDa. Binding experiments indicated high affinity for bovine placental lactogen (bPL) and human growth hormone (hGH) as compared to ovine (o) or rat PRLs. Gel filtration was used to determine the stoichiometry of rPRLR-ECDs interaction with these hormones. At a 5 microM initial concentration of the hormones, formation of 2:1 (ECD:ligand) complexes was detected with bPL, hGH and oPRL whereas only 1:1 complex was formed with rPRL. Dilution (25-fold) of these complexes did not affect the stoichiometry with bPL, whereas with hGH a clear tendency towards dissociation of the initial 2:1 complex to 1:1 complex was observed. This tendency was even stronger in the case of oPRL. Although all four hormones exhibited nearly identical activities in the Nb2-11C lymphoma cell bioassay, the ability of the purified rat or rabbit PRLR-ECD to inhibit hormonal mitogenic activity generally reflected their affinity for the respective hormones. In view of these and former results, we suggest that unlike in the GH:GHR-ECD interaction, the inability of lactogenic hormones to form a 1:2 complex with soluble recombinant PRLR-ECDs does not necessarily predicts lack of biological activity.

Interaction between the SifA virulence factor and its host target skip is essential for salmonella pathogenesis

SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a ΔsifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.

Involvement of a subset of tyrosine kinases and phosphatases in regulation of the β-lactoglobulin gene promoter by prolactin

This study used pharmacological intervention to provide support for a role of kinases and phosphatases in prolactin transactivation of a milk protein gene. It was based on transient cotransfection using a rabbit prolactin receptor expression plasmid and a beta-lactoglobulin promoter/CAT reporter construct. In cotransfected CHO cells, herbimycin A and tyrphostin, two tyrosine kinase inhibitors, were able to decrease the CAT response by over 50%, along with tyrosine phosphorylation of cellular proteins, whereas genistein and lavendustine were without effect on lactoglobulin transactivation. Orthovanadate, an inactivator of tyrosine phosphatases, was able to substitute for prolactin in inducing the CAT response. Staurosporine, a non-specific kinase inhibitor, was able, when used at low concentrations (10 nM), to augment the prolactin response strikingly. Threonine/serine kinases do not appear to be involved early in beta-lactoglobulin promoter transactivation, since four C-kinase inhibitors and okadaic acid a threonine/serine phosphatase inhibitor, were without substantive effect. We conclude that specific tyrosine kinases are responsible for most of the signal transduction from the prolactin receptor to the beta-lactoglobulin gene promoter.

Prolactin induces growth inhibition and promotes differentiation of CHO cells stably transfected with prolactin receptor complementary DNA

We have characterized a stable and functional transfectant of the rabbit prolactin receptor in Chinese hamster ovary cells, and investigated the action of prolactin (PRL) on the growth and differentiation of this transfectant (clone E32). PRL induced a significant inhibition of E32 cell proliferation. Growth inhibition correlated with gene induction of the molecular marker of ovarian differentiation cholesterol side chain cleavage P450 (P450scc). Both effects were inversely proportional to cell confluence. The limits and potential development of such transfected cellular systems are discussed.

PROLACTIN RECEPTOR AND SIGNAL TRANSDUCTION TO MILK PROTEIN GENES

Abstract After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca++ concentration. PRL stimulates Ca++ entry and induces secondary Ca++ mobilization. The entry of Ca++ is a result of an increase in K+ conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 μM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the β lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.

Interaction of lactogenic hormones with the soluble extracellular domain of prolactin receptors.

Abstract Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E. coli (amino acids 4-210) expression systems. Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E. coli expression system. All four proteins were purified as monomers with >95% homogeneity. Their affinity for various lactogenic and somatogenic hormones was determined by binding assays. The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays. The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity. This conclusion does not preclude however, hormoneor antibody-induced dimerization of the membrane-embedded receptor.