Accessible Requires Authentication Published by De Gruyter September 8, 2012

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

Vincent Gerard Francis and Sathyanarayana N. Gummadi
From the journal Biological Chemistry

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.


Corresponding author

Received: 2012-2-25
Accepted: 2012-6-4
Published Online: 2012-09-08
Published in Print: 2012-10-01

©2012 by Walter de Gruyter Berlin Boston