A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay
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; se...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2016-09-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520309408 |
| _version_ | 1818417872919068672 |
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| author | Jonathan K. Fleming Thomas R. Glass Steve J. Lackie Jonathan M. Wojciak |
| author_facet | Jonathan K. Fleming Thomas R. Glass Steve J. Lackie Jonathan M. Wojciak |
| author_sort | Jonathan K. Fleming |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T12:13:41Z |
| format | Article |
| id | doaj.art-950f5f4f9b4c469faee15971b0df0f6e |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T12:13:41Z |
| publishDate | 2016-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-950f5f4f9b4c469faee15971b0df0f6e2022-12-21T23:01:41ZengElsevierJournal of Lipid Research0022-22752016-09-0157917371747A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion AssayJonathan K. Fleming0Thomas R. Glass1Steve J. Lackie2Jonathan M. Wojciak3Lpath Inc., San Diego, CA 92121Sapidyne Instruments Inc., Boise, ID 83705Sapidyne Instruments Inc., Boise, ID 83705Lpath Inc., San Diego, CA 92121; To whom correspondence should be addressed.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.http://www.sciencedirect.com/science/article/pii/S0022227520309408anti-lipid antibodyapolipoproteinsKinetic Exclusion Assaylipidscompetitive affinity analysislipoproteins |
| spellingShingle | Jonathan K. Fleming Thomas R. Glass Steve J. Lackie Jonathan M. Wojciak A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay Journal of Lipid Research anti-lipid antibody apolipoproteins Kinetic Exclusion Assay lipids competitive affinity analysis lipoproteins |
| title | A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay |
| title_full | A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay |
| title_fullStr | A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay |
| title_full_unstemmed | A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay |
| title_short | A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay |
| title_sort | novel approach for measuring sphingosine 1 phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the kinetic exclusion assay |
| topic | anti-lipid antibody apolipoproteins Kinetic Exclusion Assay lipids competitive affinity analysis lipoproteins |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520309408 |
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