Hyaluronan synthase mediates dye translocation across liposomal membranesReport as inadecuate




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BMC Biochemistry

, 13:2

Cellular and membrane biochemistry

Abstract

BackgroundHyaluronan HA is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase HAS, which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS SeHAS to liposomes preloaded with the fluorophore Cascade Blue CB.

ResultsCB translocation efflux was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin BCL. An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin TO-CL and that, in contrast, tetramyristoyl cardiolipin TM-CL is an inactivating lipid Weigel et al, J. Biol. Chem. 281 , 36542, 2006. Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL.

ConclusionsThe results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

AbbreviationsAbantibody

BCLbovine cardiolipin

CBCascade Blue

DDMn-dodecyl-β-D-maltoside

FLfluorescence

HAhyaluronic acid: hyaluronate: hyaluronan

HASHA synthase

MDmembrane domain

MRP5multidrug resistant protein 5

NEMN-ethylmaleimide

PBSphosphate buffered saline

PEphospatidyl ethanolamine

PGphosphatidyl glycerol

PLphospholipid

SeHASStreptococcus equisimilis HAS

SpHASStreptococcus pyogenes HAS

TM-CLtetramyristoyl cardiolipin TO-CL: tetraoleoyl cardiolipin.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2091-13-2 contains supplementary material, which is available to authorized users.

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Author: Andria P Medina - Jialing Lin - Paul H Weigel

Source: https://link.springer.com/article/10.1186/1471-2091-13-2



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