Hyaluronan synthase mediates dye translocation across liposomal membranes

CB 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.The 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.Hyaluronic acid (hyaluronan; HA) is a linear unbranched polysaccharide composed of repeating disaccharide units of N-acetyl-glucosamine and D-glucuronic acid. In vertebrates and streptococcal species, HA is synthesized by Class I HA synthases (HAS), lipid-dependent integral membrane proteins with eight membrane domains (MDs) in eukaryotes or six MDs in species such as S. pyogenes and S. equisimilis [1,2]. Mammals express three HAS isozymes encoded by distinct genes designated HAS1, HAS2 and HAS3. HA is an essential glycosaminoglycan in vertebrate extracellular matrices [3], where it helps maintain the physical structure and integrity of tissues, in particular cartilage. HA is also a major constituent of skin, vitreous humor in the eye, synovial fluid in joints, and the cumulus cell matrix that s