title: Effect of a cholesterol-rich lipid environment on the enzymatic activity of reconstituted hyaluronan synthase.
abstract: Hyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-environment. The culture of human dermal fibroblasts in lipoprotein-depleted medium attenuated the synthesis of hyaluronan. The sequestration of cellular cholesterol by methyl-ß-cyclodextrin also decreased the hyaluronan production of fibroblasts, as well as the HAS activity. To directly evaluate the effects of cholesterol on HAS activity, a recombinant human HAS2 protein with a histidine-tag was expressed as a membrane protein by using a baculovirus system, then successfully solubilized, and isolated by affinity chromatography. When the recombinant HAS2 proteins were reconstituted into liposomes composed of both saturated phosphatidylcholine and cholesterol, this provided a higher enzyme activity as compared with the liposomes formed by phosphatidylcholine alone. Cholesterol regulates HAS2 activity in a biphasic manner, depending on the molar ratio of phosphatidylcholine to cholesterol. Furthermore, the activation profiles of different lipid compositions were determined in the presence or absence of cholesterol. Cholesterol had the opposite effect on the HAS2 activity in liposomes composed of phosphatidylethanolamine or phosphatidylserine. Taken together, the present data suggests a clear functional association between HAS activity and cholesterol-dependent alterations in the physical and chemical properties of cell membranes.
authors: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan.
authors: Pawared
authors: P
authors: Ontong
authors: Department of Molecular Oncology, Division of Molecular and Cellular Biology, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, Matsumoto, Nagano 390-8621, Japan.
authors: Yasuyo
authors: Y
authors: Hatada
authors: Department of Molecular Oncology, Division of Molecular and Cellular Biology, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, Matsumoto, Nagano 390-8621, Japan.
authors: Shun'ichiro
authors: S
authors: Taniguchi
authors: Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
authors: Ikuko
authors: I
authors: Kakizaki
authors: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan; Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan. Electronic address: itanon@cc.kyoto-su.ac.jp.
authors: Naoki
authors: N
authors: Itano
mesh_terms: D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D050356:Lipid Metabolism / Q000502:physiology*
chemical_list: D006820:Hyaluronic Acid; D002784:Cholesterol; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases
keywords: Biosynthesis; Cholesterol; DMPC; DMPE; DMPS; DOPC; DOPE; DOPS; DPPC; DPPE; DPPS; DSPC; DSPE; DSPS; Hyaluronan; Lipid; PC; PE; PS; Synthase; dimyristoyl PC; dimyristoyl PE; dimyristoyl PS; dioleoyl PC; dioleoyl PE; dioleoyl PS; dipalmitoyl PC; dipalmitoyl PE; dipalmitoyl PS; distearoyl PC; distearoyl PE; distearoyl PS; phosphatidylcholine; phosphatidylethanolamine; phosphatidylserine