Hyaluronan synthase mediates dye translocation across liposomal membranes

Abstract

Background: Hyaluronan (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).

Results: 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.

Conclusions: 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.

Figure 1

Experimental model for monitoring HAS-mediated FL quenching in liposomes. Step 1 in the scheme depicts the addition of purified SeHAS (cylinders) to extrusion liposomes (large circles) containing free CB (small circles). In step 2, HAS molecules insert into the liposome bilayer, allowing the small CB dye to translocate (efflux) through the HAS pores to the exterior if an intraprotein pore is present. In experiments with continuous FL monitoring in the presence of external anti-CB Ab (black Y), CB that effluxes from the lumen is bound by Ab resulting in FL quenching (step 3).

Figure 2

Incorporation of SeHAS into extrusion liposomes induces CB quenching. A. Extrusion liposomes loaded with CB were prepared, purified and stored at 4°C under argon as described in Methods. Liposomes were suspended in 0.25 ml 0.1 M NaCl, 51 mM sodium phosphate, 3.8 mM Na citrate, pH 7.4 (~50 μM PL) with 10 μg/ml anti-CB Ab and equilibrated at 30°C in cuvettes in the fluorometer. FL was monitored at 433 nm for 5 min to obtain a stable base-line (time-zero), and then SeHAS (15 μl; purified with no DDM or glycerol in the elution buffer) was added to 200 nM (black circles; mean ± SE, n = 3) or the same volume of mock-purified empty-vector membranes (white circles) or elution buffer (white squares) was added. Samples were mixed well and FL monitoring was continued. Decreasing FL (ongoing quenching) was observed after SeHAS addition, but not after addition of elution buffer or mock-purified material from empty-vector membranes. B. Purified SeHAS was added to extrusion liposomes as in A and after 30 min the suspension was centrifuged, the supernatant was saved, and the pellet was resuspended and centrifuged. The final pellet (lane 2) was resuspended to the same volume as the original supernatant (lane 3), trichloroacetic acid was added (to 10%; w/v) to both samples and precipitated protein was centrifuged, redissolved in Laemmli buffer and analyzed by SDS-PAGE and Western blotting with anti-SeHAS Ab. Lane 1 is a sample of purified SeHAS precipitated and redissolved in parallel as a control.

Figure 3

SeHAS-dependent quenching in extrusion liposomes is concentration dependent. Different amounts of purified SeHAS, in the same volume of Elution Buffer, were added to CB-containing liposomes at 30°C to final concentrations of 50 (black squares), 100 (black triangles), or 200 (black circles) nM and FL changes were recorded as in Fig 2. In this and the following Figs, FL quenching (1.0 - relative FL) is graphed, rather than relative FL. After 1800 seconds, quenching was 3%, 6%, and 14% in response to addition of 50, 100 and 200 nM SeHAS, respectively.

Figure 4

SeHAS mediates greater CB release from extrusion liposomes containing activating TO-CL compared to inhibitory TM-CL. Purified SeHAS was added to liposomes containing (mol%) 70% PE, 25% PG and either 5% TO-CL (black circles) or TM-CL (black triangles). Subsequent release of CB and FL quenching by anti-CB Ab was monitored as described in Fig 2. Values are the mean ± SE (n = 3). Note that the FL quenching scale is greater than in other Figs.

Figure 5

SeHAS inactivated by heat, oxidation or NEM modification does not mediate CB release from extrusion liposomes. A. Heat inactivation. CB-containing liposomes were treated at 30°C as in Fig 2 except that before addition to liposomes, purified SeHAS was either treated at 47°C for 5 min to inactivate the enzyme (black triangles) or left untreated on ice (black circles). FL quenching was monitored as in Fig 2. B. Oxidative inactivation. Purified oxidized SeHAS was prepared by omitting DTT in the elution buffer. Oxidized SeHAS was then either left oxidized (black triangles) or was reduced by treatment with 1 mM DTT for 1 hr at 4°C (black circles) and then added to extrusion liposomes and FL quenching monitored as in Fig 2. The response with reduced and rescued SeHAS is essentially identical to the untreated enzyme in A. C. NEM modification. Purified SeHAS was untreated (black circles) or treated (black triangles) for 15 min at 4°C with 6 mM NEM, which inhibits SeHAS activity (24); DTT (12 mM) was then added to react with the remaining NEM. SeHAS was added to CB-liposomes to a final concentration of 200 nM and FL changes were monitored as in Fig 2. CB-liposomes without SeHAS were also incubated in the same way with NEM and DTT as a control (black squares) to assess possible leakage.

Figure 6

K48E and K48F mutants mediate less CB release from liposomes than WT SeHAS. Purified WT (black circles) and K48E (Panel A, black squares) or K48F (Panel B, black squares) SeHAS were added to CB-containing liposomes and FL quenching was monitored at 30°C for the indicated times as in Fig. 2.