Internalization of OspA in rsCD14 complex and aggregated forms.
Although the spirochetal protein OspA is capable of stimulating immune cells in a CD14- and TLR2-dependent manner, little is known about how TLR2 receptor complex ligands, such as OspA, are handled by the cell once delivered. We examine here the internalization of the fluorescently derivatized forms of both the full length OspA lipoprotein delivered as a recombinant soluble CD14 (rsCD14) complex and the corresponding lipohexapeptide given to the cells as an aggregate. Both forms of OspA are internalized in a similar manner to acetylated low density lipoprotein (AcLDL), a scavenger receptor ligand. Acetylated low density lipoprotein is capable of competing for internalization with OspA even when OspA is delivered as a rsCD14 complex. We observe co-localization of OspA with lysosomes but not with the Golgi complex. These phenomena are similar between RAW264.7 macrophages and endothelial cells but change drastically when the cells are deprived of serum. Upon serum starvation, OspA shows some localization to the Golgi apparatus whereas the lipohexapeptide remains on the cell surface. Inhibition of internalization of OspA via treatment with cytochalasin D or of the lipohexapeptide via serum starvation does not interfere with TNF induction activity, consistent with signalling from the cell surface.
Welty
Diane M
DM
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S 4th St. Hamilton, MT 59840, USA.
Snyder
D Scott
DS
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D000954:Antigens, Surface / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001428:Bacterial Vaccines; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D002518:Ceramides; D016895:Culture Media, Serum-Free; D018950:Lipopolysaccharide Receptors; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C072045:OspA protein; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D015638:Cytochalasin D; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan