Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4)

Abstract

Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca²⁺, Mg²⁺, and Zn²⁺ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca²⁺ and Mg²⁺ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca²⁺ has more affinity than Mg²⁺ and Zn²⁺. Stains-all studies further confirm that hPLSCR4 is a Ca²⁺-binding protein. Point mutation (Asp²⁹⁰→Ala) in hPLSCR4 decreased the Ca²⁺-binding affinity as well as Tb³⁺ luminescence, suggesting residues of the predicted Ca²⁺-binding motif are involved in Ca²⁺ binding. Functional reconstitution with (Asp²⁹⁰→Ala) mutant led to ∼50% and ∼40% decrease in scramblase activity in the presence of Ca²⁺ and Mg²⁺, respectively.