title: Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications.
abstract: Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.
authors.affiliation: Biomolecular Science Center, University of Central Florida, Orlando, Florida 32826, USA.
authors.forename: Abhay H
authors.initials: AH
authors.lastname: Pande
authors.forename: Shan
authors.initials: S
authors.lastname: Qin
authors.forename: Kathleen N
authors.initials: KN
authors.lastname: Nemec
authors.forename: Xiaomei
authors.initials: X
authors.lastname: He
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
mesh_terms: D000818:Animals; D001514:Bee Venoms / Q000201:enzymology; D054501:Group II Phospholipases A2; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000737:chemistry; D008564:Membrane Potentials; D008566:Membranes / Q000201:enzymology; D008958:Models, Molecular; D010741:Phospholipases A / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D001514:Bee Venoms; D007527:Isoenzymes; D008563:Membrane Lipids; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2