Hyaluronan synthase mediates dye translocation across liposomal membranes

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.

Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X7-B motif

“…The original proposal that HAS translocates the growing HA-UDP chain through an intra-protein pore (Tlapak-Simmons et al 1999a) has been verified (Hubbard et al 2012;Medina et al 2012). We now understand that HAS enzymes function either as monomers (Tlapak-Simmons et al 1998) or oligomers (Bart et al 2015) and their processive mechanism utilizes an intra-HAS pore or deep protein cleft (Kumari et al 2006) within the plasma membrane ( Figure 7) that serves simultaneously to retain and to translocate the growing HA-UDP chain to the cell exterior (Hubbard et al 2012;Medina et al 2012;Bi et al 2015;Weigel 2015). Many questions still remain regarding the mechanism of synthesis and what causes and controls HA release and HA product size.…”

Cancer may be a pathway to cell survival under persistent hypoxia and elevated ROS: A model for solid‐cancer initiation and early development

“…Note that the synthesis of hyaluronic acid is done through repeated addition of one glucuronic acid and one N ‐acetylglucosamine to the nascent polydisaccharide as the molecule is extruded via ABC transporters or hyaluronic acid synthase into the extracellular space . The exported hyaluronic acid will be degraded by some of the hyaluronidases ( HYAL1–6 ) or by ROS into fragments of different sizes if it is not incorporated into extracellular matrices …”

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

“…The relatively small size and few transmembrane domains of streptococcal HAS suggest that they cannot facilitate pore formation for translocation of the growing HA chain (Weigel, 2002). However, recent work showed that HAS derived from S. equisimilis (SeHAS; encoded by seHas) could mediate the release of the small fluorescent dye Cascade Blue from inside liposomes, and that mutations, which decreased HAS activity and HA MW in an earlier study, in one of its transmembrane domains resulted in reduced Cascade Blue efflux (Kumari, Baggenstoss, Parker, & Weigel, 2006;Medina, Lin, & Weigel, 2012). However, recent work showed that HAS derived from S. equisimilis (SeHAS; encoded by seHas) could mediate the release of the small fluorescent dye Cascade Blue from inside liposomes, and that mutations, which decreased HAS activity and HA MW in an earlier study, in one of its transmembrane domains resulted in reduced Cascade Blue efflux (Kumari, Baggenstoss, Parker, & Weigel, 2006;Medina, Lin, & Weigel, 2012).…”