A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

Abstract

(GPCRs) (1-3). Many physiological processes, such as cell growth, differentiation, survival, motility, and angiogenesis (3), and pathophysiological processes, such as cancer, cardiovascular disease, multiple sclerosis, neuropathic pain, and fibrosis (4, 5), 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 (1,4,6). 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) and reportedly binds 50-70% of plasma S1P, whereas serum albumin reportedly binds 30% or more (8-10) . 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). Human plasma contains approximately 0.9 M apoM (11, 12), where >95% of the total apoM occupies 5% of the HDL (apoM-HDL) and <2% of the LDL (apoM-LDL) in plasma (13,14); this stoichiometry results in less than 1 mol of S1P per mole of HDL in human plasma (15). S1P-associated HDL stimulates cellular 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 (K d ) for these carrier proteins binding S1P and the major LPA species. Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lysophospholipids that bind and signal through multiple G protein-coupled receptors

Determinants of Serum- and Plasma Sphingosine-1-Phosphate Concentrations in a Healthy Study Group

“…This scenario is supported by the fact that albumin binds S1P with very low affinity as the dissociation constant for albumin is more than 3 orders of magnitude higher when compared with HDL or LDL. 28 Serum-S1P concentrations correlate with total cholesterol and LDL-C, but not with HDL-C and the same pattern is also observed for plasma-S1P, though with lower correlation-coefficients (►Fig. 3B).…”

High‐density lipoprotein from end‐stage renal disease patients exhibits superior cardioprotection and increase in sphingosine‐1‐phosphate

“…Whilst plasma S1P is principally associated with HDL, also albumin serves as a transport vector. The higher affinity of S1P for HDL compared to albumin explains the distribution towards HDL in plasma. This would be exacerbated if a lower level of albumin were available in the circulation to compete for HDL in plasma.…”

A Novel Perspective on the ApoM-S1P Axis, Highlighting the Metabolism of ApoM and Its Role in Liver Fibrosis and Neuroinflammation

“…Thus, apoM secreted by hepatocytes [ 21 ] or resident in blood plasma [ 12 ] correlates with plasma S1P levels [ 22 ]. Human serum albumin (SA) contains 3 binding sites for long-chain fatty acids [ 23 ] and binds S1P with a K D of ~22 µM [ 24 ]. ApoM-deficient mice display 50% reduced plasma S1P levels, no detectable S1P in HDL, and unchanged S1P levels in the albumin fraction [ 12 ].…”

“…This supports the hypothesis that apoM bound S1P is actively utilized, while albumin rather serves as a reservoir and scavenger for free S1P [ 12 , 25 ]. Interestingly, S1P shows a significantly lower binding affinity to HDL associated apoM (K D 21 nM) as to LDL bound apoM (K D 2.4 nM) [ 24 ] but also with a binding affinity significantly lower than previously reported for recombinant apoM [ 8 ]. Thus, a conformational change mediated by the lipoprotein phospholipid layer may enhance S1P recognition and binding to apoM.…”

“…Albumin reabsorption in the proximal tubule is mediated by cubilin, a co-receptor interacting with megalin [ 64 ]. It can however be only speculated whether albumin acts as an alternative S1P scavenger, notably due to its low S1P binding affinity [ 24 ] and putative occupation by other molecules. Renal uptake of apoM [ 50 ] and albumin [ 63 , 65 ] is probably accompanied by either lysosomal degradation and consequent release of scavenged S1P, or export to peritubular capillaries.…”