title: Targeting of scavenger receptor class B type I by synthetic amphipathic alpha-helical-containing peptides blocks lipopolysaccharide (LPS) uptake and LPS-induced pro-inflammatory cytokine responses in THP-1 monocyte cells.
abstract: Human scavenger receptor class B type I, CLA-1, mediates lipopolysaccharide (LPS) binding and internalization (Vishnyakova, T. G., Bocharov, A. V., Baranova, I. N., Chen, Z., Remaley, A. T., Csako, G., Eggerman, T. L., and Patterson, A. P. (2003) J. Biol. Chem. 278, 22771-22780). Because one of the recognition motifs in SR-B1 ligands is the anionic amphipathic alpha-helix, we analyzed the effects of model amphipathic alpha-helical-containing peptides on LPS uptake and LPS-stimulated cytokine production. The L-37pA model peptide, containing two class A amphipathic helices, bound with high affinity (K(d) = 0.94 microg/ml) to CLA-1-expressing HeLa cells with a 10-fold increased capacity when compared with mock transfected HeLa cells. Both LPS and L-37pA colocalized with anti-CLA-1 antibody and directly bound CLA-1 as determined by cross-linking. SR-BI/CLA-1 ligands such as HDL, apoA-I, and L-37pA efficiently competed against iodinated L-37pA. Bacterial LPS, lipoteichoic acid, and hsp60 also competed against iodinated L-37pA. Model peptides blocked uptake of iodinated LPS in both mock transfected and CLA-1-overexpressing HeLa cells. Bound and internalized Alexa-L-37pA and BODIPY-LPS colocalized at the cell surface and perinuclear compartment. Both ligands were predominantly transported to the Golgi complex, colocalizing with the Golgi markers bovine serum albumin-ceramide, anti-Golgin97 antibody, and cholera toxin subunit B. A 100-fold excess of L-37pA nearly eliminated BODIPY-LPS binding and internalization. L-37pA and its d-amino acid analogue, D-37pA peptide were similarly effective in blocking LPS, Gram-positive bacterial wall component lipoteichoic acid and bacterial heat shock protein Gro-EL-stimulated cytokine secretion in THP-1 cells. In the same culture media used for the cytokine stimulation study, neither L-37pA nor D-37pA affected the Limulus amebocyte lysate activity of LPS, indicating that LPS uptake and cytokine stimulation were blocked independently of LPS neutralization. These results demonstrate that amphipathic helices of exchangeable apolipoproteins may represent a general host defense mechanism against inflammation.
authors.affiliation: Department of Laboratory Medicine, W. G. Magnuson Clinical Center, NIDDK, National Institutes of Health. Bethesda, Maryland 20892, USA. abocharov@mail.cc.nih.gov
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Bocharov
authors.forename: Irina N
authors.initials: IN
authors.lastname: Baranova
authors.forename: Tatyana G
authors.initials: TG
authors.lastname: Vishnyakova
authors.forename: Alan T
authors.initials: AT
authors.lastname: Remaley
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Csako
authors.forename: Fairwell
authors.initials: F
authors.lastname: Thomas
authors.forename: Amy P
authors.initials: AP
authors.lastname: Patterson
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Eggerman
mesh_terms: D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry* / Q000378:metabolism; D018955:CD36 Antigens; D002417:Cattle; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D051116:Receptors, Scavenger; D051117:Scavenger Receptors, Class A; D051122:Scavenger Receptors, Class B; D013682:Teichoic Acids / Q000378:metabolism
chemical_list: D001053:Apolipoproteins; D018955:CD36 Antigens; D016207:Cytokines; D008070:Lipopolysaccharides; D010455:Peptides; D011971:Receptors, Immunologic; D051116:Receptors, Scavenger; C495124:SCARB1 protein, human; D051117:Scavenger Receptors, Class A; D051122:Scavenger Receptors, Class B; D013682:Teichoic Acids; C009900:lipoteichoic acid