title: 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.
authors.affiliation: Department of Biotechnology, Applied Industrial Microbiology Laboratory, Indian Institute of Technology-Madras, Chennai 600 036, India.
authors.forename: Vincent Gerard
authors.initials: VG
authors.lastname: Francis
authors.forename: Sathyanarayana N
authors.initials: SN
authors.lastname: Gummadi
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; C493425:PLSCR4 protein, human; D049294:Phospholipid Transfer Proteins; D002118:Calcium