Regulation of P-selectin glycoprotein ligand 1 (PSGL-1) interactions with selectins : role of the decameric repeats and of the cytoplasmic domain

SUMMARY BACKGROUND: P-selectin glycoprotein ligand 1 (PSGL-1) is a major selectin ligand, mediating leukocyte rolling along inflamed vascular wall. It is a mucin-like homodimer composed of a N-terminal domain which binds selectins, followed by 14-16 decameric repeats (DR), a transmembrane domain and a cytoplasmic tail, which may be involved in regulating leukocyte rolling and in generating intracellular signals, through its binding to moesin and Syk. P- and L-selectin binding is dependent on core-2 O-glycosylation and tyrosine sulfation of PSGL-1 N-terminus. However, a minor part of E-selectin-mediated rolling is dependent on N-terminal O-glycans; additional binding sites may thus be involved. In this project, we studied whether (1) PSGL-1 DR and (2) PSGL-1 cytoplasmic residues which bind moesin, were also involved in the regulation of selectin-dependent rolling. METHODS: Several mutated cDNAs were obtained: (1) PSGL-1 DR were either deleted, or substituted by platelet GPlba macroglycopeptide, (2) Ser-336, -348, Lys-337 and Arg-338 were mutated to alanine; moreover, truncation mutants retaining only 6 or 2 cytoplasmic residues were also generated. Transfected CHO expressing mutant PSGL-1 were tested for their ability to bind soluble selectin chimeras and to support selectin-dependent rolling under flow conditions. RESULTS: (1) Deletion of the DR had a dramatic effect on P- and L-selectin-dependent cell recruitment and rolling stability, which could only partially be compensated for, by GPlba substitution. In addition, we observed that DR create a binding site for E-selectin and thus support PSGL-1-dependent rolling. (2) Flow assays revealed that the moesin-binding site, in particular Ser-336, plays a crucial role in regulating the recruitment, velocity and rolling stability of PSGL-1-expressing cells on P- and L-selectin. CONCLUSIONS: Data presented here highlight the structure -function relationship of PSGL-1 DR. Moreover, they reveal a crucial role for the moesin-binding residues in regulating P-and L-selectin-dependent rolling. RÉSUMÉ CONTEXTE: PSGL-1 (P-selectin glycoprotein ligand 1) est un ligand majeur des sélectines permettant le roulement des leucocytes le long de la paroi vasculaire enflammée. C'est un homodimère de type mucine, composé d'un domaine N-terminal liant les sélectines, suivi de 14-16 répétitions décamèriques (RD), d'un domaine transmembranaire et d'une queue cytoplasmique qui pourrait être impliquée dans la régulation du roulement leucocytaire et la génération de signaux intracellulaires, via sa liaison à la moésine et à Syk. La liaison à la Pet à la L-sélectine dépend de la présentation par le N-terminus de PSGL-1 de O-glycans sur des structures core-2 et de tyrosines sulfatées. Cependant, une fraction mineure du roulement médié par la E-sélectine dépend des O-glycans N-terminaux; des sites de liaisons supplémentaires pourraient donc être impliqués. Dans ce projet, nous avons étudié si (1) les RD de PSGL-1 ainsi que (2) les résidus cytoplasmiques liant la moésine, étaient impliqués dans la régulation du roulement dépendant des sélectines. MÉTHODES: Plusieurs ADN codant des formes mutées de PSGL-1 ont été obtenus: (1) Les RD de PSGL-1 ont été soit ôtées, soit remplacées par le macroglycopeptide de la GPlba plaquettaire, (2) les Ser-336, -348, la Lys-337 et l'Arg-338 ont été mutées en alanine; par ailleurs, des mutants tronqués ne retenant plus que 6 ou 2 résidus cytoplasmiques ont également été générés. Des CHO transfectées exprimant PSGL-1 muté ont été testées pour leur capacité à lier des sélectines chimériques solubles et à soutenir un roulement dépendant des sélectines dans des conditions de flux. RÉSULTATS: (1) La perte des RD a eu un effet dramatique sur le recrutement cellulaire et la stabilité de roulement dépendant des P- et L-sélectine, qui n'a pu être que partiellement compensé par la substitution par la GPlba. De plus, nous avons observé que les RD forment un site de liaison pour la E-sélectine et soutiennent ainsi le roulement dépendant de PSGL-1. (2) Les tests de flux ont révélé que le site de liaison à la moésine, notamment la Ser-336, joue un rôle crucial dans la régulation du recrutement, de la vitesse et de la stabilité du roulement des cellules exprimant PSGL-1 sur les P- et L-sélectine. CONCLUSIONS; Les données présentées ici ont permis d'éclaircir la relation structure -fonction des RD de PSGL-1. Par ailleurs, elles révèlent un rôle crucial pour les résidus liant la moésine dans le roulement dépendant des P- et L-sélectine. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC Pour accomplir ses fonctions, le sang circule sur un réseau de 96'000 kilomètres; ainsi, il approvisionne les cellules de l'organisme en énergie, il transporte diverses substances, il assure la défense contre les pathogènes et il participe à la régulation de la température corporelle. Le sang contient plusieurs types de cellules: la grande majorité sont les globules rouges, auxquels il faut ajouter les plaquettes (dont le rôle est de colmater les lésions vasculaires) et les globules blancs (leucocytes) qui, bien que présents en très faible quantité (moins de 0.01 %), jouent un rôle crucial en cas d'infection ou d'inflammation. Une attaque par un pathogène provoque plusieurs changements (rougeur, chaleur, gonflement, douleur), qui sont des manifestations de l'inflammation. Pour atteindre l'agent infectieux, des globules blancs spécialisés (les granulocytes) doivent quitter la circulation sanguine. Afin de faciliter leur capture, les vaisseaux sanguins vont exprimer des protéines telles que les sélectines, qui sont reconnues par une protéine leucocytaire appelée PSGL-1 (P-selectin glycoprotein ligand 7). L'interaction des sélectines avec PSGL-1 soutient le roulement du globule blanc le long de la paroi vasculaire, à une vitesse très inférieure à celle du flux sanguin. Ce roulement conduit à l'activation du globule blanc par des molécules de l'inflammation, permettant son adhésion ferme, puis son arrêt. Finalement, le granulocyte va migrer à travers la paroi du vaisseau pour atteindre et éliminer les causes de l'inflammation. L'adhésion est un processus intéressant à caractériser, car outre l'inflammation, il est également impliqué dans l'artériosclérose, l'infarctus, la métastatisation et la thrombose. Dans ce travail, nous nous sommes intéressés à définir les rôles des différents domaines de PSGL-1 dans la régulation de son interaction avec les sélectines. En effet, en plus de son extrémité extracellulaire de haute affinité pour les sélectines, PSGL-1 est composé de plusieurs séquences répétées hautement glycosylées et d'une courte région intracellulaire, dont les fonctions n'avaient pas été étudiées auparavant. En créant des formes mutées de PSGL-1, nous avons pu montrer qu'un roulement efficace des leucocytes nécessite la présence des régions répétitives et du domaine intracellulaire au complet.

An essential role for the stalk region of CD8 beta in the coreceptor function of CD8.

The CD8alphabeta heterodimer is integral to the selection of the class I-restricted lineage in the thymus; however, the contribution of the CD8beta chain to coreceptor function is poorly understood. To understand whether the CD8beta membrane proximal stalk region played a role in coreceptor function, we substituted it with the corresponding sequence from the CD8alpha polypeptide and expressed the hybrid molecule in transgenic mice in place of endogenous CD8beta. Although the stalk-swapped CD8beta was expressed on the cell surface as a disulfide-bonded heterodimer at equivalent levels of expression to an endogenous CD8beta molecule, it failed to restore selection of CD8(+) class I MHC-restricted T cells and it altered the response of peripheral T cells. Thus, the stalk region of the CD8beta polypeptide has an essential role in ensuring functionality of the CD8alphabeta heterodimer and its replacement compromises the interaction of CD8 with peptide-MHC complexes.

Differential expression of NLRP3 among hematopoietic cells.

Although the importance of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in health and disease is well appreciated, a precise characterization of NLRP3 expression is yet undetermined. To this purpose, we generated a knock-in mouse in which the Nlrp3 coding sequence was substituted for the GFP (enhanced GFP [egfp]) gene. In this way, the expression of eGFP is driven by the endogenous regulatory elements of the Nlrp3 gene. In this study, we show that eGFP expression indeed mirrors that of NLRP3. Interestingly, splenic neutrophils, macrophages, and, in particular, monocytes and conventional dendritic cells showed robust eGFP fluorescence, whereas lymphoid subsets, eosinophils, and plasmacytoid dendritic cells showed negligible eGFP levels. NLRP3 expression was highly inducible in macrophages, both by MyD88- and Trif-dependent pathways. In vivo, when mice were challenged with diverse inflammatory stimuli, differences in both the number of eGFP-expressing cells and fluorescence intensity were observed in the draining lymph node. Thus, NLRP3 levels at the site of adaptive response initiation are controlled by recruitment of NLRP3-expressing cells and by NLRP3 induction.

Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.

Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the cross-recognition.

Basic calcium phosphate crystals induce IL-1B secretion in vitro through activation of the Nalp3 inflammasome

Objectives: We tested the effects of the three forms of basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbonate-substituted apatite (CA) and hydroxyapatite (HA)) on monocytes and macrophages on IL-1β secretion. The requirement for the NALP3 inflammasome and TLR2 and TLR4 receptors in this acute response was analyzed.

De Novo Production of Metabolites by Fungal Co-culture of Trichophyton rubrum and Bionectria ochroleuca.

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.

Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the alpha1B-adrenergic receptor.

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the alpha1B-adrenergic receptor (alpha1BAR). In this study, phosphoamino acid analysis of the phosphorylated alpha1BAR revealed that both epinephrine- and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist- as well as phorbol ester-induced phosphorylation and desensitization of the alpha1BAR. To identify the serines invoved in agonist- versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Our findings indicate that Ser394 and Ser400 were phosphorylated following phorbol ester-induced activation of protein kinase C, whereas Ser404, Ser408, and Ser410 were phosphorylated upon stimulation of the alpha1BAR with epinephrine. The observation that overexpression of G protein-coupled kinase 2 (GRK2) could increase agonist-induced phosphorylation of Ser404, Ser408, and Ser410, strongly suggests that these serines are the phosphorylation sites of the alpha1BAR for kinases of the GRK family. Phorbol ester-induced phosphorylation of the Ser394 and Ser400 as well as GRK2-mediated phosphorylation of the Ser404, Ser408, and Ser410, resulted in the desensitization of alpha1BAR-mediated inositol phosphate response. This study provides generalities about the biochemical mechanisms underlying homologous and heterologous desensitization of G protein-coupled receptors linked to the activation of phospholipase C.

Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity.

Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin-containing cytoplasmic focal accumulations of the substituted protein to a degree that was correlated with the severity of the associated phenotypes. In contrast, study of mutations leading to substitutions in the FLNB rod domain that result in the same phenotypes as ABD mutations demonstrated that with only one exception disease-associated substitutions, surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism.