Abstract
Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membrane-disrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.
Highlights
Non-trypanolytic high density lipoprotein (HDL), i.e. HDL depleted of Trypanosome lytic factor (TLF) by passage over an ␣-haptoglobinrelated protein (Hpr) affinity resin, did not exhibit permeabilizing activity against these particular liposomes at any pH (Fig. 1A and data not shown)
To determine the nature of the particle size changes revealed by light scattering, we performed transmission electron microscopy on negatively stained preparations of liposomes treated with TLF, Hpr, apolipoprotein L-1 (apoL-1), or apolipoprotein A-1 (apoA-1) (Fig. 5, B–G)
The ultimate step in trypanosome killing by TLF is permeabilization of the lysosomal membrane. We have addressed this particular step with the use of simplified model liposomes, a technique utilized to study myriad membrane-interacting proteins and peptides, including human HDL
Summary
Lipids and Reagents—All lipids were purchased from Avanti Polar Lipids (Alabaster, AL). A non-trypanolytic fraction of human HDL was prepared from the unbound material passaged over the anti-Hpr immunoaffinity chromatography (Ͼ99% of the total human HDL). This HDL fraction was depleted in Hpr and lacked trypanosome-killing activity. Assays were performed by diluting liposomes 1:1000 into the appropriate buffer and monitoring fluorescence with an LS-55 spectrofluorometer from PerkinElmer Life Sciences. Assays were performed by incubating 100 g of lipid with 2.5 g of TLF in a final volume of 50 l at 37 °C for 30 min in the indicated buffer. Electron Microscopy—Unilamellar liposomes were constructed from soy bean azolectin and prepared by extrusion (0.1 m). Samples were adsorbed to glow-discharged Formvar-coated copper grids, stained with 2% uranyl acetate, and imaged with a FEI Tecnai 20 transmission electron microscope
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