Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease

Sphingosine 1-phosphate (S1P) is a bioactive signaling lipid highly enriched in mature erythrocytes. Previous study has revealed that levels of S1P are significantly elevated in patients and mice with Sickle Cell Disease (SCD), a devastating and highly prevalent genetic hemolytic disorder that causes life-threatening hemolysis, tissue damage, and organ dysfunction with very limited treatment. Moreover, the activity of S1P generating enzyme-Sphingosine Kinase 1 (SphK1) is increased in human and mouse SCD erythrocytes, and inhibition of SphK1 decreased erythrocyte sickling. However, the structural and functional basis for the pathogenic nature of S1P in SCD remains obscure. Here, we report that increased erythrocyte S1P promotes pathogenic metabolic reprogramming coupled to increased channeling of glucose to glycolysis rather than through the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increasing deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, hemolysis and disease progression. S1P functioning intracellularly binds to deoxyHbS, facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the PPP. Extending from SCD, we unexpectedly found that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure of HbA complexed with S1P alone or in combination with 2,3-BPG at 1.9 Å resolution revealed the overall architecture and unique features of S1P-2,3-BPG-deoxyHbA complex. In the presence of 2,3-BPG, S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to a1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with b2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying pathogenic consequences of elevated S1P in SCD and its potential role in normal erythrocyte physiology.