FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2476205345> ?p ?o ?g. }

• W2476205345 endingPage "1747" @default.
• W2476205345 startingPage "1737" @default.
• W2476205345 abstract "Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling. Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling. Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lysophospholipids that bind and signal through multiple G protein-coupled receptors (GPCRs) (1.Kihara Y. Maceyka M. Spiegel S. Chun J. Lysophospholipid receptor nomenclature review: IUPHAR Review 8.Br. J. Pharmacol. 2014; 171: 3575-3594Crossref PubMed Scopus (223) Google Scholar, 2.Mutoh T. Rivera R. Chun J. Insights into the pharmacological relevance of lysophospholipid receptors.Br. J. Pharmacol. 2012; 165: 829-844Crossref PubMed Scopus (114) Google Scholar, 3.Chun J. Lysophospholipid Receptors: Signaling and Biochemistry. Wiley, Hoboken, NJ.. Wiley, Hoboken, NJ2013Crossref Scopus (3) Google Scholar). Many physiological processes, such as cell growth, differentiation, survival, motility, and angiogenesis (3.Chun J. Lysophospholipid Receptors: Signaling and Biochemistry. Wiley, Hoboken, NJ.. Wiley, Hoboken, NJ2013Crossref Scopus (3) Google Scholar), and pathophysiological processes, such as cancer, cardiovascular disease, multiple sclerosis, neuropathic pain, and fibrosis (4.Kihara Y. Mizuno H. Chun J. Lysophospholipid receptors in drug discovery.Exp. Cell Res. 2015; 333: 171-177Crossref PubMed Scopus (142) Google Scholar, 5.Tigyi G. Aiming drug discovery at lysophosphatidic acid targets.Br. J. Pharmacol. 2010; 161: 241-270Crossref PubMed Scopus (137) Google Scholar), involve S1P or LPA signaling. The S1P and LPA pathways are validated therapeutic targets; many drugs and pharmacological agents have been developed to modulate the activity of receptors and enzymes in these pathways (1.Kihara Y. Maceyka M. Spiegel S. Chun J. Lysophospholipid receptor nomenclature review: IUPHAR Review 8.Br. J. Pharmacol. 2014; 171: 3575-3594Crossref PubMed Scopus (223) Google Scholar, 4.Kihara Y. Mizuno H. Chun J. Lysophospholipid receptors in drug discovery.Exp. Cell Res. 2015; 333: 171-177Crossref PubMed Scopus (142) Google Scholar, 6.Ishii I. Fukushima N. Ye X. Chun J. Lysophospholipid receptors: signaling and biology.Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (647) Google Scholar). Many of these compounds block circulating S1P and LPA from binding and activating cognate membrane-bound receptors. Circulating S1P exists primarily bound to carrier molecules, including HDL, LDL, and serum albumin. HDL is a protein-rich lipoprotein containing multiple protein constituents (7.Xu N. Dahlback B. A novel human apolipoprotein (apoM).J. Biol. Chem. 1999; 274: 31286-31290Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) and reportedly binds ∼50–70% of plasma S1P, whereas serum albumin reportedly binds ∼30% or more (8.Murata N. Sato K. Kon J. Tomura H. Yanagita M. Kuwabara A. Ui M. Okajima F. Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions.Biochem. J. 2000; 352: 809-815Crossref PubMed Scopus (357) Google Scholar, 9.Schuchardt M. Tolle M. Prufer J. van der Giet M. Pharmacological relevance and potential of sphingosine 1-phosphate in the vascular system.Br. J. Pharmacol. 2011; 163: 1140-1162Crossref PubMed Scopus (60) Google Scholar, 10.Hammad S.M. Al Gadban M.M. Semler A.J. Klein R.L. Sphingosine 1-phosphate distribution in human plasma: associations with lipid profiles.J. Lipids. 2012; 2012: 180705Crossref PubMed Google Scholar). apoM represents the main protein component in HDL responsible for binding S1P, and the X-ray cocrystal structure of recombinant human apoM in complex with S1P has been solved (11.Christoffersen C. Obinata H. Kumaraswamy S.B. Galvani S. Ahnstrom J. Sevvana M. Egerer-Sieber C. Muller Y.A. Hla T. Nielsen L.B. et al.Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M.Proc. Natl. Acad. Sci. USA. 2011; 108: 9613-9618Crossref PubMed Scopus (446) Google Scholar). Human plasma contains approximately 0.9 μM apoM (11.Christoffersen C. Obinata H. Kumaraswamy S.B. Galvani S. Ahnstrom J. Sevvana M. Egerer-Sieber C. Muller Y.A. Hla T. Nielsen L.B. et al.Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M.Proc. Natl. Acad. Sci. USA. 2011; 108: 9613-9618Crossref PubMed Scopus (446) Google Scholar, 12.Axler O. Ahnstrom J. Dahlback B. An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma.J. Lipid Res. 2007; 48: 1772-1780Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), where >95% of the total apoM occupies ∼5% of the HDL (apoM-HDL) and <2% of the LDL (apoM-LDL) in plasma (13.Arkensteijn B.W. Berbee J.F. Rensen P.C. Nielsen L.B. Christoffersen C. The apolipoprotein m-sphingosine-1-phosphate axis: biological relevance in lipoprotein metabolism, lipid disorders and atherosclerosis.Int. J. Mol. Sci. 2013; 14: 4419-4431Crossref PubMed Scopus (31) Google Scholar, 14.Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. Isolation and characterization of human apolipoprotein M-containing lipoproteins.J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar); this stoichiometry results in less than 1 mol of S1P per mole of HDL in human plasma (15.Kontush A. Lhomme M. Chapman M.J. Unraveling the complexities of the HDL lipidome.J. Lipid Res. 2013; 54: 2950-2963Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). S1P-associated HDL stimulates cellular pathways that promote endothelial barrier function, suggesting that S1P mediates the protective effects of HDL against atherosclerosis (16.Argraves K.M. Gazzolo P.J. Groh E.M. Wilkerson B.A. Matsuura B.S. Twal W.O. Hammad S.M. Argraves W.S. High density lipoprotein-associated sphingosine 1-phosphate promotes endothelial barrier function.J. Biol. Chem. 2008; 283: 25074-25081Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). While S1P bound apoM-HDL suppresses vascular inflammation, S1P delivered using albumin did not show this effect in vitro, suggesting divergent roles for S1P chaperones in maintaining the vasculature and other physiological processes (17.Galvani S. Sanson M. Blaho V.A. Swendeman S.L. Obinata H. Conger H. Dahlback B. Kono M. Proia R.L. Smith J.D. et al.HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation.Sci. Signal. 2015; 8 ([Erratum. 2015. Sci. Signal. 8: er8.]): ra79Crossref PubMed Scopus (214) Google Scholar, 18.Blaho V.A. Galvani S. Engelbrecht E. Liu C. Swendeman S.L. Kono M. Proia R.L. Steinman L. Han M.H. Hla T. HDL-bound sphingosine-1-phosphate restrains lymphopoiesis and neuroinflammation.Nature. 2015; 523: 342-346Crossref PubMed Scopus (158) Google Scholar). In blood, LPA also exists bound to carrier proteins, primarily serum albumin (19.Tigyi G. Henschen A. Miledi R. A factor that activates oscillatory chloride currents in Xenopus oocytes copurifies with a subfraction of serum albumin.J. Biol. Chem. 1991; 266: 20602-20609Abstract Full Text PDF PubMed Google Scholar, 20.Tigyi G. Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochromocytoma cells.J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar). Total LPA in plasma comprises several distinct species, which contain esterified fatty acids with varying numbers of carbon atoms and cis double bonds (Fig. 1A) capable of activating cognate GPCRs with varying potencies (21.Yung Y.C. Stoddard N.C. Chun J. LPA receptor signaling: pharmacology, physiology, and pathophysiology.J. Lipid Res. 2014; 55: 1192-1214Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 22.Bandoh K. Aoki J. Taira A. Tsujimoto M. Arai H. Inoue K. Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structure-activity relationship of cloned LPA receptors.FEBS Lett. 2000; 478: 159-165Crossref PubMed Scopus (223) Google Scholar). Although albumin is the most abundant protein in human plasma and LPA is one of the first bioactive lipids identified, the stoichiometry and mechanism of interaction between these two molecules is poorly understood. As with fatty acids, serum albumin has the capacity to bind multiple LPA molecules per protein molecule (23.Kragh-Hansen U. Watanabe H. Nakajou K. Iwao Y. Otagiri M. Chain length-dependent binding of fatty acid anions to human serum albumin studied by site-directed mutagenesis.J. Mol. Biol. 2006; 363: 702-712Crossref PubMed Scopus (82) Google Scholar, 24.Richieri G.V. Anel A. Kleinfeld A.M. Interactions of long-chain fatty acids and albumin: determination of free fatty acid levels using the fluorescent probe ADIFAB.Biochemistry. 1993; 32: 7574-7580Crossref PubMed Scopus (336) Google Scholar, 25.Bhattacharya A.A. Grune T. Curry S. Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin.J. Mol. Biol. 2000; 303: 721-732Crossref PubMed Scopus (742) Google Scholar). Studies suggest that albumin contains three strong affinity long-chain fatty acid binding sites, and these are the same sites occupied by LPA (26.Thumser A.E. Voysey J.E. Wilton D.C. The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin.Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (117) Google Scholar, 27.Bojesen I.N. Bojesen E. Albumin binding of long-chain fatty acids: thermodynamics and kinetics.J. Phys. Chem. 1996; 100: 17981-17985Crossref Scopus (31) Google Scholar). The method described in this report uses monoclonal antibodies (mAbs) to compete with purified serum albumin or isolated lipoprotein particles for binding S1P and LPA in solution (see cartoon schematic in Fig. 1B). The production and characterization of the two humanized IgG1k mAbs, LT1009 and LT3015, 3LT3015 and LT1009 are proprietary agents of Lpath Inc., but may be provided upon request. which specifically recognize S1P and LPA, respectively, and the structural basis for lipid recognition are described elsewhere (28.Wojciak J.M. Zhu N. Schuerenberg K.T. Moreno K. Shestowsky W.S. Hiraiwa M. Sabbadini R. Huxford T. The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen.Proc. Natl. Acad. Sci. USA. 2009; 106: 17717-17722Crossref PubMed Scopus (43) Google Scholar, 29.Fleming J.K. Wojciak J.M. Campbell M.A. Huxford T. Biochemical and structural characterization of lysophosphatidic acid binding by a humanized monoclonal antibody.J. Mol. Biol. 2011; 408: 462-476Crossref PubMed Scopus (18) Google Scholar). These antibodies directly compete with carrier proteins for binding target lipids in vitro; the equilibrium binding curve for LT3015 binding LPA shifts toward weaker apparent affinity as the concentration of fatty acid-free (FAF)-BSA is increased (Fig. 1C). During competition binding, the equilibrium dissociation constants (Kd) for the antibody-lipid and protein-lipid interactions govern the concentration of antigen-free binding site on the antibody. Therefore, measuring the free antibody enables the Kd for both the antibody (Kd1) and the serum protein (Kd2) binding S1P or LPA to be determined. The Kinetic Exclusion Assay (KinExA®) is a technique for measuring the Kd of protein-ligand interactions through direct detection of the ligand-free binding sites in a sample. This technique is compatible with a variety of biological systems and has several advantages over other methods (30.Darling R.J. Brault P.A. Kinetic exclusion assay technology: characterization of molecular interactions.Assay Drug Dev. Technol. 2004; 2: 647-657Crossref PubMed Scopus (68) Google Scholar, 31.Blake 2nd, R.C. Pavlov A.R. Blake D.A. Automated kinetic exclusion assays to quantify protein binding interactions in homogeneous solution.Anal. Biochem. 1999; 272: 123-134Crossref PubMed Scopus (117) Google Scholar, 32.Bee C. Abdiche Y.N. Stone D.M. Collier S. Lindquist K.C. Pinkerton A.C. Pons J. Rajpal A. Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.PLoS One. 2012; 7: e36261Crossref PubMed Scopus (26) Google Scholar, 33.Drake A.W. Tang M.L. Papalia G.A. Landes G. Haak-Frendscho M. Klakamp S.L. Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex.Anal. Biochem. 2012; 429: 58-69Crossref PubMed Scopus (66) Google Scholar). KinExA is particularly attractive to study protein-lipid interactions because native untagged molecules binding entirely in solution can be investigated. Using modified (non-native) lipid molecules or covalently attaching bulky tags or fluorophores may significantly alter the solubility properties of the lipids or the mechanisms of protein recognition. To overcome these issues, we developed a label-free method for determining the Kd for S1P and LPA binding carrier proteins in solution. Using KinExA and simultaneously fitting several competitive curves (competition n-curve analysis), we measured the Kd values for FAF-BSA and FAF-human serum albumin (HSA) binding S1P and five predominant individual LPA species: palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and arachidonic (20:4). Because only the Kd value for LPA(18:1) binding albumin has been reported (34.Goetzl E.J. Lee H. Azuma T. Stossel T.P. Turck C.W. Karliner J.S. Gelsolin binding and cellular presentation of lysophosphatidic acid.J. Biol. Chem. 2000; 275: 14573-14578Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 35.Ojala P.J. Hermansson M. Tolvanen M. Polvinen K. Hirvonen T. Impola U. Jauhiainen M. Somerharju P. Parkkinen J. Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine.Biochemistry. 2006; 45: 14021-14031Crossref PubMed Scopus (50) Google Scholar), we were encouraged to measure the Kd values for other biologically active LPA species to investigate whether LPA species with different numbers of carbon atoms or unsaturated bonds in the acyl chain show different affinities for serum albumin. Indeed, we observed an ∼70-fold difference in Kd values between LPA(16:0) and LPA(20:4) binding FAF-HSA. We also used this method, along with a published apoM ELISA (36.Bosteen M.H. Dahlback B. Nielsen L.B. Christoffersen C. Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA.J. Lipid Res. 2015; 56: 754-759Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), to investigate S1P binding apoM in the context of isolated human HDL and LDL particles. Sevvana et al. (37.Sevvana M. Ahnstrom J. Egerer-Sieber C. Lange H.A. Dahlback B. Muller Y.A. Serendipitous fatty acid binding reveals the structural determinants for ligand recognition in apolipoprotein M.J. Mol. Biol. 2009; 393: 920-936Crossref PubMed Scopus (57) Google Scholar) showed that S1P quenched the intrinsic fluorescence of purified recombinant human apoM with an IC50 value of 0.9 μM. This value is ∼45-fold and ∼375-fold higher (weaker binding) than the Kd values reported here for S1P binding to apoM-HDL and apoM-LDL, respectively. In addition, S1P appears to bind apoM-LDL with significantly stronger affinity compared with apoM-HDL, suggesting mechanistic differences in the apoM-S1P interaction between these lipoprotein particles. Finally, the Kd values for LT1009 and LT3015 binding their cognate lipid antigens absent the effects of the carrier proteins used to deliver the lipids have been determined. The use of carrier proteins to deliver lysophospholipids is ubiquitous in lipid research. Here, we demonstrate that the binding affinities of certain carrier proteins (or chaperones) for S1P or LPA are significant, and these interactions likely influence the bioavailability of the lipid and activation of their cognate receptors. The antigen-free binding sites on LT1009 and LT3015 were measured using a KinExA 3200 equipped with an autosampler. The antibody was captured using modified S1P and LPA, which contain a mercapto group covalently attached to the omega carbon atom and cross-linked to maleimide-activated BSA (Thermo Scientific) (3.Chun J. Lysophospholipid Receptors: Signaling and Biochemistry. Wiley, Hoboken, NJ.. Wiley, Hoboken, NJ2013Crossref Scopus (3) Google Scholar). The purified S1P-BSA and LPA-BSA conjugates were diluted (30 μg/ml) with PBS without calcium and magnesium (PBS, Cellgro), and 1 ml of the solution was added to 0.2 g of PMMA beads (Sapidyne Instruments Inc.). The slurry was rocked for 1 h at 37°C to adsorb the conjugate onto the beads. After coating, the beads were blocked with 150 μM Fraction V FAF-BSA (Calbiochem) in PBS. The concentrations of the protein stock solutions were determined by measuring the absorbance at 280 nm and using an extinction coefficient of 1.4 ml/mg for LT1009 and LT3015 and 0.66 ml/mg for FAF-BSA and FAF-HSA (Sigma-Aldrich) (38.Yarmush D.M. Morel G. Yarmush M.L. A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy.J. Biochem. Biophys. Methods. 1987; 14: 279-289Crossref PubMed Scopus (14) Google Scholar, 39.Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. How to measure and predict the molar absorption coefficient of a protein.Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3437) Google Scholar, 40.Esposito B.P. Faljoni-Alario A. de Menezes J.F. de Brito H.F. Najjar R. A circular dichroism and fluorescence quenching study of the interactions between rhodium(II) complexes and human serum albumin.J. Inorg. Biochem. 1999; 75: 55-61Crossref PubMed Scopus (32) Google Scholar, 41.Shpak A.P. Gorbik P.P. Nanomaterials and Supramolecular Structures: Physics, Chemistry, and Applications.. Springer, Dordrecht, London, New York2009Google Scholar). S1P, LPA(16:0), LPA(18:0), LPA(18:1), and LPA(20:4) (Avanti Polar Lipids) and LPA(18:2) (Echelon Biosciences) were resuspended in methanol by repeated sonication and vortex mixing. The lysophospholipid concentration in each stock solution was determined using a colorimetric total phosphorus assay using a protocol from Avanti Polar Lipids, which was based on work by Chen, Toribara, and Huber (42.Chen P.S. Toribara T.Y. Huber W. Microdetermination of phosphorus.Anal. Chem. 1956; 28: 1756-1758Crossref Scopus (5834) Google Scholar) and Fiske and Subbarow (43.Fiske C.H. Subbarow Y. The colorimetric determination of phosphorus.J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar). Aliquots of the resuspended lipid stocks were transferred to glass vials via a glass syringe and the solvent was evaporated under a dry argon stream. In order to demonstrate that the experiments described below were conducted in the so-called “KinExA mode,” i.e., dissociation of antibody-lipid complexes in solution does not significantly contribute to the antibody captured on the solid phase, we monitored the percentage of free antibody while systematically increasing the flow rate (32.Bee C. Abdiche Y.N. Stone D.M. Collier S. Lindquist K.C. Pinkerton A.C. Pons J. Rajpal A. Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.PLoS One. 2012; 7: e36261Crossref PubMed Scopus (26) Google Scholar). The percentage of free antibody is calculated by dividing the signal (voltage) of the antibody in the presence of the target lipid by the signal of the antibody in the absence of the lipid (signal at 100% free antibody) after correcting for nonspecific binding. At equilibrium, samples containing the following antibody/lipid/albumin concentrations yielded approximately 50% free antibody signal at the slowest flow rate (0.25 ml/min): 1) 10 nM LT3015, 13 nM LPA(16:0), and 13 nM FAF-BSA; 2) 10 nM LT3015, 65 nM LPA(20:4), and 13 nM FAF-BSA; and 3) 10 nM LT1009, 14 nM S1P, and 1 μM FAF-BSA. As the flow rate increased, the percentage of free antibody in these solutions does not change significantly (supplemental Fig. S1), demonstrating that the fraction of antibody in complex with the lipid does not contribute to the free antibody measurements in the equilibrium affinity experiments. Dried aliquots of S1P were resuspended in S1P running buffer [10 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2, 0.005% polysorbate 20, 0.02 NaN3 (pH 7.4)] containing 100 μM FAF-BSA by sonication and vortex mixing to yield a 0.1 mM S1P stock solution. Calcium was included in the running buffer because divalent metal ions bridge the antibody-S1P interface and are required for strong affinity binding (28.Wojciak J.M. Zhu N. Schuerenberg K.T. Moreno K. Shestowsky W.S. Hiraiwa M. Sabbadini R. Huxford T. The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen.Proc. Natl. Acad. Sci. USA. 2009; 106: 17717-17722Crossref PubMed Scopus (43) Google Scholar). A series of 2-fold S1P dilutions were prepared in glass vials using a glass syringe using the running buffer above with 100 μM FAF-BSA. For LPA experiments, dried LPA aliquots were resuspended in PBS containing 15 μM FAF-BSA by sonication and vortex mixing to yield LPA stock solutions of 0.5 mM [LPA(16:0), LPA(18:0), LPA(18:1), LPA(18:2)] or 5 mM [LPA(20:4)], and a series of 2-fold dilutions were prepared using a glass syringe with PBS plus 15 μM FAF-BSA. During all these preparations, extra precautions were taken to minimize lipid material loss, and the syringes were washed several times between titrations to minimize lipid carry-over. For each equilibrium affinity experiment, the antibody and serum albumin concentrations were constant. For S1P experiments, samples containing either 1 or 10 nM LT1009 and 1 or 500 μM FAF-BSA (dependent on experiment) in S1P running buffer were prepared in silanized glass tubes (Thermo Scientific). For each of the LPA species, samples containing either 0.5 or 10 nM LT3015 IgG and 13 nM or 10 μM FAF-BSA (dependent on experiment) in PBS were prepared in silanized glass tubes. S1P or LPA was added to the antibody and FAF-BSA-containing tubes from the titrated lipid stocks described above using a glass syringe from low to high lipid concentration to minimize lipid carry-over. FAF-HSA was used in place of FAF-BSA for the 10 nM LT1009, 500 μM FAF-HSA and the 10 nM LT3015, 10 μM FAF-HSA experiments. Sample sets containing antibody, serum albumin, and titrated lipid were allowed to equilibrate (6–24 h depending on experiment) prior to data collection. Flow rates of 2.25 ml/min and 0.25 ml/min for LPA and S1P experiments, respectively, were used and shown to kinetically exclude dissociation of antibody-lipid complexes during capture of the free antibody. The captured antibody was detected using a goat anti-human Alexa Fluor or DyLight secondary (Jackson ImmunoResearch). The free antibody in each sample was measured in duplicate. Data were analyzed using drift correction in the competition n-curve software (Sapidyne Instruments); see details in the Data analysis procedures section. HDL (d 1.063–1.21 g/ml) and LDL (d 1.019–1.063 g/ml) isolated from a single normal human male donor using KBr ultracentrifugation and gel filtration chromatography (HDL only) were purchased from EMD Millipore and used as lipoprotein stock solutions without further purification. Two equilibrium affinity experiments were set up where endogenous S1P in the HDL and LDL stock solutions served as the sole ligand/antigen source. Here, 2-fold serial dilutions of the HDL or LDL stocks (starting at 1:10 HDL or 1:6.1 LDL) containing either 1 or 10 nM LT1009 were prepared in S1P running buffer supplemented with 1 μM FAF-BSA. Three additional sample sets containing a constant amount of HDL or LDL and a 2-fold serial titration of exogenous S1P were prepared. Again, S1P was delivered via glass syringe from low to high lipid concentration to samples containing either 1 or 10 nM LT1009 and a fixed dilution of the lipoprotein stock (HDL, 1:80, 1:450, and 1:1,200; LDL, 1:40, 1:225, and 1:600). The free LT1009 in each fraction was measured in duplicate, and the data were analyzed using drift correction in the competition n-curve software as detailed in Data analysis procedures below. The binding experiments described here involved a carrier protein (FAF-BSA, FAF-HSA, apoM-HDL, or apoM-LDL) in the reaction mixture. Generally, the Kd for the binding of the lipid to the carrier protein is more than an order of magnitude weaker than the antibody binding, however experimental conditions often dictate the carrier protein be present at concentrations more than an order of magnitude higher than the antibody. This results in a situation where the antibody and carrier protein compete for the lipid, an effect which must be taken into account when measuring the Kd values of the antibody-lipid and carrier protein-lipid interactions. The mathematics of competitive binding is well-known and both exact and implicit solutions to the fundamental equations of binding and conservation of mass have been described (44.Wang Z.X. An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule.FEBS Lett. 1995; 360: 111-114Crossref PubMed Scopus (289) Google Scholar, 45.Thomä N. Goody R.S. What to do if there is no signal: using competition experiments to determine binding parameters..In Kinetic Analysis of Macromolecules: A Practical Approach. K. A. Johnson, editor. Oxford University Press, Oxford, UK. 2003; : 153-170Google Scholar). In the present case, we implemented an implicit solution of the following set of equations: Kd1 = [Ab][L]/[AbL], Kd2 = [P][L]/[PL], [Ab]T = [Ab] + [AbL], [P]T = [P] + [PL], and [L]T = [L] + [AbL] + [PL]; where Kd1 and Kd2 are the equilibrium binding constants of lipid (L) for antibody (Ab) and carrier protein (P), respectively. The final three equations express conservation of mass requirements for binding. In the present analyses, the total concentrations of the antibody, [Ab]T, and the carrier protein, [P]T, were treated as independent variables. The concentration of free antibody, [Ab], was measured using the KinExA instrument. The Kd values and total lipid concentration, [L]T, were varied to minimize the least squared error to the measured data. Uniqueness of fit was assessed by construction of error curves for the fitted parameters. After optimum values of the parameters had been determined, one parameter at a time was moved away from its optimum value and the remaining parameters were reoptimized; the residual error was plotted versus the varying parameter. Presence of a distinct minima in the resulting error graph is treated as evidence that the optimized value was unique (i.e., all other values of this parameter resulted in a worse fit to the measured data). Achieving unique fits for the pa" @default.
• W2476205345 created "2016-08-23" @default.
• W2476205345 creator A5001402848 @default.
• W2476205345 creator A5013427525 @default.
• W2476205345 creator A5017277144 @default.
• W2476205345 creator A5020194935 @default.
• W2476205345 date "2016-09-01" @default.
• W2476205345 modified "2023-10-03" @default.
• W2476205345 title "A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay" @default.
• W2476205345 cites W1519679921 @default.
• W2476205345 cites W1531820450 @default.
• W2476205345 cites W1549006420 @default.
• W2476205345 cites W1566845866 @default.
• W2476205345 cites W1569922252 @default.
• W2476205345 cites W1664264312 @default.
• W2476205345 cites W1905243785 @default.
• W2476205345 cites W1970976992 @default.
• W2476205345 cites W1974608867 @default.
• W2476205345 cites W1980078657 @default.
• W2476205345 cites W1982033373 @default.
• W2476205345 cites W1985105527 @default.
• W2476205345 cites W1985342306 @default.
• W2476205345 cites W1985551319 @default.
• W2476205345 cites W1986426214 @default.
• W2476205345 cites W1988129027 @default.
• W2476205345 cites W1990875180 @default.
• W2476205345 cites W1992495544 @default.
• W2476205345 cites W1998134241 @default.
• W2476205345 cites W2006736579 @default.
• W2476205345 cites W2012785373 @default.
• W2476205345 cites W2018046936 @default.
• W2476205345 cites W2019889454 @default.
• W2476205345 cites W2023797086 @default.
• W2476205345 cites W2029417795 @default.
• W2476205345 cites W2032511565 @default.
• W2476205345 cites W2037890379 @default.
• W2476205345 cites W2046958785 @default.
• W2476205345 cites W2049869221 @default.
• W2476205345 cites W2057633574 @default.
• W2476205345 cites W2061731390 @default.
• W2476205345 cites W2061822447 @default.
• W2476205345 cites W2063790487 @default.
• W2476205345 cites W2065094485 @default.
• W2476205345 cites W2067547649 @default.
• W2476205345 cites W2070382695 @default.
• W2476205345 cites W2072708194 @default.
• W2476205345 cites W2087767169 @default.
• W2476205345 cites W2089475778 @default.
• W2476205345 cites W2089751595 @default.
• W2476205345 cites W2095103144 @default.
• W2476205345 cites W2104017463 @default.
• W2476205345 cites W2108598678 @default.
• W2476205345 cites W2108812575 @default.
• W2476205345 cites W2119812858 @default.
• W2476205345 cites W2124440700 @default.
• W2476205345 cites W2129214913 @default.
• W2476205345 cites W2129351604 @default.
• W2476205345 cites W2135805245 @default.
• W2476205345 cites W2137735884 @default.
• W2476205345 cites W2139432714 @default.
• W2476205345 cites W2147589893 @default.
• W2476205345 cites W2153435210 @default.
• W2476205345 cites W2157438880 @default.
• W2476205345 cites W2159801302 @default.
• W2476205345 cites W2162705046 @default.
• W2476205345 cites W2165183251 @default.
• W2476205345 cites W2167270736 @default.
• W2476205345 cites W2184796395 @default.
• W2476205345 cites W2261586976 @default.
• W2476205345 cites W2952533352 @default.
• W2476205345 cites W974691727 @default.
• W2476205345 doi "https://doi.org/10.1194/jlr.d068866" @default.
• W2476205345 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5003157" @default.
• W2476205345 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27444045" @default.
• W2476205345 hasPublicationYear "2016" @default.
• W2476205345 type Work @default.
• W2476205345 sameAs 2476205345 @default.
• W2476205345 citedByCount "21" @default.
• W2476205345 countsByYear W24762053452017 @default.
• W2476205345 countsByYear W24762053452018 @default.
• W2476205345 countsByYear W24762053452019 @default.
• W2476205345 countsByYear W24762053452020 @default.
• W2476205345 countsByYear W24762053452021 @default.
• W2476205345 countsByYear W24762053452022 @default.
• W2476205345 countsByYear W24762053452023 @default.
• W2476205345 crossrefType "journal-article" @default.
• W2476205345 hasAuthorship W2476205345A5001402848 @default.
• W2476205345 hasAuthorship W2476205345A5013427525 @default.
• W2476205345 hasAuthorship W2476205345A5017277144 @default.
• W2476205345 hasAuthorship W2476205345A5020194935 @default.
• W2476205345 hasBestOaLocation W24762053451 @default.
• W2476205345 hasConcept C153911025 @default.
• W2476205345 hasConcept C159654299 @default.
• W2476205345 hasConcept C16613235 @default.
• W2476205345 hasConcept C170493617 @default.
• W2476205345 hasConcept C185592680 @default.
• W2476205345 hasConcept C203014093 @default.
• W2476205345 hasConcept C2776596873 @default.

• next