title: Interaction of peptide-bound beads with lipopolysaccharide and lipoproteins.
abstract: We previously reported the generation of lipopolysaccharide (LPS)-binding peptides by phage display and chemical modification. Among them, a dodecapeptide designated Li5-025 (K'YSSSISSIRAC'; K' and C' denote d-lysine and d-cysteine, respectively) showed a high binding affinity for LPS and was resistant to protease digestion (Suzuki et al., 2010). In the current study, Li5-025-bound silica beads, hereafter referred to as P-beads, were generated and found to be devoid of LPS-neutralizing activity. Thus, LPS bound to the P-beads could be directly used in the Limulus amebocyte lysate (LAL) assay. P-beads bound LPS dissolved in solutions of ethanol, pH4, pH10, and 0.5M NaCl and LPS bound to the P-beads was quantitatively assayed. The sensitivity of this assay was observed to be approximately 0.1pg/mL LPS. P-beads bound LPS dissolved in antithrombin III (AT III) solution which is a strong inhibitor of activated factors C and B as well as the clotting enzyme in the LAL assay; the inhibitory effect of AT III was completely reversed upon washing the P-beads with 25% acetonitrile. This was employed as the first step for the detection of free LPS in plasma using the LAL assay. LPS added to human plasma at 0°C followed by application to the P-beads and subsequent washing with 25% acetonitrile resulted in low LPS activity as detected by the LAL assay. However, further washing of the P-beads with 0.1% Triton X100 in 25% acetonitrile resulted in high LPS activity. This is the first instance of quantitative detection of free LPS in plasma using the LAL assay, and the sensitivity of this method was observed to be 1pg/mL of LPS. The proteins eluted in the 0.1% Triton X-100 wash were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two protein bands of 28kDa and 18kDa were predominantly observed. Mass spectrometry analysis revealed that the 28kDa and 18kDa bands corresponded to apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), respectively. ApoA-I and apoA-II are components of high density lipoprotein (HDL). Thus, it is likely that the P-beads-bound LPS was sequestered by HDL, resulting in neutralization of its toxicity. This study showed that by using P-beads, free LPS in plasma can be quantitatively measured by the LAL assay at a concentration of 1pg/mL.
authors.affiliation: Peptide Door Co. Ltd., Fukushima-machi 794-21, Takasaki, Gunma 370-3523, Japan.
authors.forename: Masatsugu M
authors.initials: MM
authors.lastname: Suzuki
authors.affiliation: Peptide Door Co. Ltd., Fukushima-machi 794-21, Takasaki, Gunma 370-3523, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Peptide Door Co. Ltd., Fukushima-machi 794-21, Takasaki, Gunma 370-3523, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Omi
authors.affiliation: Peptide Door Co. Ltd., Fukushima-machi 794-21, Takasaki, Gunma 370-3523, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Graduate School of Medicine, Gunma University, Showa-machi 3-39-15, Maebashi, Gunma 371-8511, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: Department of Molecular Physiology and Medical Bioregulation, Graduate School of Medicine, Yamaguchi University, Minami-Kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kishi
authors.affiliation: Department of Molecular Physiology and Medical Bioregulation, Graduate School of Medicine, Yamaguchi University, Minami-Kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Sei
authors.initials: S
authors.lastname: Kobayashi
authors.affiliation: Peptide Door Co. Ltd., Fukushima-machi 794-21, Takasaki, Gunma 370-3523, Japan. Electronic address: pep-takagi@peptide-door.com.
authors.forename: Takashi
authors.initials: T
authors.lastname: Takagi
mesh_terms: D001774:Blood Chemical Analysis; D006801:Humans; D008033:Limulus Test / Q000379:methods*; D008070:Lipopolysaccharides / Q000032:analysis*; D008074:Lipoproteins / Q000378:metabolism*; D008863:Microspheres; D010455:Peptides / Q000378:metabolism*; D010949:Plasma / Q000737:chemistry*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature
chemical_list: D008070:Lipopolysaccharides; D008074:Lipoproteins; D010455:Peptides; D012965:Sodium Chloride
keywords: Antithrombin III; ApoA-I; ApoA-II; HDL; LPS