Károly Liliom

Identification of Edg1 receptor residues that recognize sphingosine 1-Phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg120 and Arg292 ion pair with the phosphate, whereas the acidic Glu121 residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [35S]GTPγS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D‐erythro‐sphingosyl‐phosphorylcholine; SPC) and sphingosine 1‐phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D‐erythro‐(2S,3R)‐SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure‐activity relationship: it requires an unsubstituted amino group because N‐acetyl‐SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein‐coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

Growth factor-like phospholipids generated after corneal injury.

The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors.

Sphingosine‐1‐phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G‐protein‐coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca2+ in intact cells independently of S1P‐GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca2+ concentration in HEK‐293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca2+ transients induced by photolysis of caged S1P were caused by Ca2+ mobilization from thapsigargin‐sensitive stores. These results provide direct evidence for a true intracellular action of S1P.

Dual coding in alternative reading frames correlates with intrinsic protein disorder

Numerous human genes display dual coding within alternatively spliced regions, which give rise to distinct protein products that include segments translated in more than one reading frame. To resolve the ensuing protein structural puzzle, we identified 67 human genes with alternative splice variants comprising a dual-coding region at least 75 nucleotides in length and analyzed the structural status of the protein segments they encode. The inspection of their amino acid composition and predictions by the IUPred and PONDR® VSL2 algorithms suggest a high propensity for structural disorder in dual-coding regions. In the case of +1 frameshifts, the average level of disorder in the two frames is similarly high (47.2% in the ancestral frame, 58.2% in the derived frame, with the average level of disorder in human proteins being approximately 30%), whereas in the case of -1 frameshifts, there is a significant tendency to become more disordered upon shifting the frame (16.7% in the ancestral frame, 56.3% in the derived frame). The regions encoded by the derived frame are mostly disordered (disorder percentage > 50%) in 39 out of 62 cases, which strongly suggests that structural disorder enables these protein products to exist and function without the need of a highly evolved 3D fold. The potential advantages are also demonstrated by the appearance of novel functions and the high incidence of transcripts escaping nonsense-mediated decay. By discussing several examples, we demonstrate that dual coding may be an effective mechanism for the evolutionary appearance of novel intrinsically disordered regions with new functions.

Identification of a Novel Growth Factor-like Lipid, 1-O-cis-Alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (Alkenyl-GP) That Is Present in Commercial Sphingolipid Preparations

Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1H and13C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 μmwas only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065–C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.

Identification of the Hydrophobic Ligand Binding Pocket of the S1P1 Receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.

Neonatal FcR Overexpression Boosts Humoral Immune Response in Transgenic Mice

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.

Stereochemical properties of lysophosphatidic acid receptor activation and metabolism

Ligand recognition by G protein-coupled receptors (GPCR), as well as substrate recognition by enzymes, almost always shows a preference for a naturally occurring enantiomer over the unnatural one. Recognition of lysophosphatidic acid (LPA) by its receptors is an exception, as both the natural L (R) and unnatural D (S) stereoisomers of LPA are equally active in bioassays. In contrast to the enantiomers of LPA, analogs of N-acyl-serine phosphoric acid (NASPA) and N-acyl-ethanolamine phosphoric acid (NAEPA), which contain a serine and an ethanolamine backbone, respectively, in place of glycerol, are recognized in a stereoselective manner. This stereoselective interaction may lead to the development of receptor subtype-selective antagonists. In the present study, we review the stereochemical aspects of LPA pharmacology and describe the chemical synthesis of pure LPA enantiomers together with their ligand-binding properties toward the LPA1, LPA2, and LPA3 receptors and their metabolism by lipid phosphate phosphatase 1 (LPP1). Finally, we evaluate the concept of stereopharmacology in developing novel ligands for LPA receptors.