Are cysteine residues of human phospholipid scramblase 1 essential for Pb²⁺ and Hg²⁺ binding-induced scrambling of phospholipids?

Abstract

Lead and mercury being common environmental pollutants are often associated with erythrocytes, where phosphatidylserine (PS) exposure-mediated procoagulant activation is induced. Human phospholipid scramblase 1 (hPLSCR1) identified in the erythrocyte membrane is a type II transmembrane protein involved in Ca²⁺-dependent bidirectional scrambling of phospholipids (PL) during blood coagulation, cell activation, and apoptosis. The prominent role of hPLSCR1 in Pb²⁺ and Hg²⁺ poisoning was demonstrated by a biochemical assay, where recombinant hPLSCR1 induced PL scrambling across bilayer with a higher binding affinity (K_d) towards Hg²⁺ (4.1 µM) and Pb²⁺ (5.8 µM) than Ca²⁺ (25.6 mM). The increased affinity could be the outcome of heavy metals interacting at auxiliary sites other than the calcium-binding motif of hPLSCR1. Similar to other metal-binding proteins, cysteine-based metal-binding motifs could be the potential additional binding sites in hPLSCR1. To explore the hypothesis, the cysteines were chemically modified, which significantly reduced only the Hg²⁺- and Pb²⁺-induced scrambling activity leaving Ca²⁺-induced activity unaltered. Recombinant constructs with deletion of prominent cysteine residues and point mutation in the calcium-binding motif including Δ100-hPLSCR1, Δ160-hPLSCR1, and D275A-hPLSCR1 were generated, purified, and assayed for scramblase activity. The cysteine-deleted constructs of hPLSCR1 showed reduced binding affinity (K_d) for Hg²⁺ and Pb²⁺ without altering the Ca²⁺-binding affinity whereas the point mutant had completely lost its affinity for Ca²⁺ and reduced affinities for Hg²⁺ and Pb²⁺. The results accentuated the significance of cysteine residues as additional binding sites for heavy metal ions in hPLSCR1.