Biotinylated peptides substituted with D-amino acids with high stability as anti-anaphylactic agents targeting platelet-activating factor.

Platelet-activating factor (PAF) is an important lipid mediator of anaphylaxis and therefore can be an anti-anaphylactic agent target. Recently, we reported that several synthetic biotinylated peptides containing a Tyr-Lys-Asp-Gly sequence markedly inhibited the bioactivities of PAF in vitro and in vivo; it also inhibited anaphylactic reactions such as hypothermia, hypotension, and vascular permeability in vivo. Here, we report the anti-anaphylactic effects of three biotinylated heptapeptides (peptide 1: H-Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, peptide 2: H-D -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, and peptide 3: H-D -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-D -Asp-OH). The experiment using tryptophan fluorescence spectroscopy showed that the interaction of peptides 2 and 3 with PAF was larger than that of peptide 1. Experiments using a rat model of hind paw edema showed that peptides 1, 3, and 2 inhibited PAF-induced edema by 67.9%, 69.3%, and 79.3%, respectively. In a mouse model of anaphylaxis, both peptides 2 and 3 showed inhibitory effects on anaphylactic hypothermia, whereas peptide 1 did not. Furthermore, experiments involving in vitro rat plasma stability of peptides showed that both peptides 3 and 2 were more stable in plasma compared to peptide 1 (84.0%, 51.8%, and 0%, remained after 6 h, respectively). Our results suggest that both peptides 2 and 3 may show systemic and local inhibitory effects as anti-anaphylactic agents targeting PAF.

Estrogen Sulfotransferase is Highly Expressed in Vascular Endothelial Cells Overlying Atherosclerotic Plaques.

Cytosolic estrogen sulfotransferase (SULT1E1) mainly catalyzes the sulfoconjugation and deactivation of estrogens that are known to exert potent anti-atherogenic effects. However, it remains unknown about the connection between SULT1E1 and atherosclerosis. Recently, we reported that SULT1E1 is highly expressed in the aorta with plaques of high fat-fed ApoE knockout (KO) mice (mouse model of atherosclerosis), and interacts with oxidized low-density lipoprotein (Ox-LDL) known as a major component of atherosclerotic lesions. In this study, immunohistochemical staining for SULT1E1 in the aorta of high fat-fed ApoE KO mice showed that SULT1E1 is detected in vascular endothelial cells overlying atherosclerotic plaques. Results from Western blotting showed that Ox-LDL induces the protein expression of both SULT1E1 and peroxisome proliferator-activated receptor (PPAR) γ in human umbilical vein endothelial cells (HUVECs), and then that a PPARγ antagonist GW9662, but not a PPARα antagonist GW6471, inhibited the protein expression of SULT1E1 induced by Ox-LDL. Moreover, GW9662 significantly increased the proliferation of HUVECs induced by Ox-LDL. Our results suggest that SULT1E1 and PPARγ, both of which are increased by Ox-LDL, may interact with each other, and then may reduce cooperatively Ox-LDL-induced proliferation of vascular endothelial cells overlying atherosclerotic plaques, leading to against atherosclerosis.

Royal Jelly Proteins Inhibit Macrophage Proliferation: Interactions with Native- and Oxidized-Low Density Lipoprotein.

Macrophage proliferation is known to correlate with macrophage accumulation in atherosclerotic plaque, and therefore its inhibition and secondary reduction of plaque inflammation may have therapeutic beneficial effects on atherosclerosis. Recently, we reported that a peptide corresponding to positions 41-51 of royalisin (which consists of 51 amino acid residues), a potent antibacterial protein contained in royal jelly (RJ), can specifically bind to oxidized LDL (Ox-LDL), a major components of atherosclerotic lesions. Here, we investigated the interaction of RJ proteins including royalisin with LDL and Ox-LDL. Measurement of LDL oxidation by the production of thiobarbituric acid reactive substances and conjugated dienes, and by electrophoretic mobility on polyacrylamide gel electrophoresis showed that RJ proteins including royalisin and the degradation products of major RJ protein (MRJP) 1 and MRJP3 can induce oxidation of LDL and Ox-LDL. Surface plasmon resonance experiments showed that these RJ proteins can exhibit much higher binding affinity to LDL than Ox-LDL (the equilibrium dissociation constant, KD = 8.35 and 49.65 µg proteins/mL for LDL and Ox-LDL, respectively). Experiments using cultured mouse J774A.1 macrophage cells proved that these RJ proteins can inhibit macrophage proliferation markedly and concentration-dependently, regardless of the absence or presence of LDL and Ox-LDL, but hardly affect lipid accumulation in macrophages. These results suggest that RJ proteins including royalisin and degradation products of MRJP1/MRJP3 may have therapeutic beneficial effects on atherosclerosis owing to the reduction of plaque inflammation. Further studies of these RJ proteins may lead to the discovery of novel anti-atherosclerotic drugs.

A biotinylated peptide, BP21, as a novel potent anti-anaphylactic agent targeting platelet-activating factor.

Platelet-activating factor (PAF) is an important mediator of anaphylaxis and is therefore an anti-anaphylactic drug target. We recently reported that synthetic N-terminally biotinylated peptides (BP4-BP29) inhibit PAF by directly interacting with PAF and its metabolite/precursor lyso-PAF. In this study, we investigated whether the biotinylated peptides can inhibit anaphylactic reactions in vivo. In mouse models of anaphylaxis, one of the peptides, BP21, markedly and dose-dependently inhibited hypothermia with a maximum dose-response within 30 min after administration, even at doses 20 times lesser than doses of the known PAF antagonist CV-3988. In contrast, the anti-hypothermic effect of BGP21, in which the Tyr-Lys-Asp-Gly sequence in BP21 was modified to a Gly-Gly-Gly-Gly sequence, was less than that of BP21. The alanine scanning and shuffling the amino acid residues of BP4 (Tyr-Lys-Asp-Gly) demonstrated that the Tyr-Lys-Asp-Gly consensus sequence is important for the inhibitory effect of the peptide on hypothermia. BP21 also suppressed vascular permeability during anaphylaxis with a maximum dose-response within 30 min of administration. In a rat model of hind paw oedema, BP21 significantly inhibited the oedema induced by PAF but not that induced by the other pro-inflammatory mediators, such as histamine, serotonin, and bradykinin. Tryptophan fluorescence measurements showed that BP21 interacted with PAF, but not with histamine, serotonin, or bradykinin. In contrast, BGP21 did not interact with PAF. These results suggest that biotinylated peptides, especially BP21, can specifically and markedly inhibit anaphylactic reactions in vivo and that this involves direct interaction of its Tyr-Lys-Asp-Gly region with PAF. Therefore, a biotinylated peptide, BP21, can be used as novel potential anti-anaphylactic drugs targeting PAF. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein.

Two oxidized forms of low-density lipoprotein (LDL), oxidized LDL (ox-LDL) and minimally modified LDL (MM-LDL), are believed to play a major role in the pathogenesis of atherosclerosis. Recently, we reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminus Lys to fluorescein isothiocyanate, (FITC)KP6, bound to ox-LDL but not to LDL. In the present study, we investigated whether (FITC)KP6 could be used as a fluorescent probe for the specific detection of MM-LDL and ox-LDL. Results from polyacrylamide gel electrophoresis and surface plasmon resonance proved that (FITC)KP6 could efficiently bind to MM-LDL as well as ox-LDL in a dose-dependent manner and with high affinity (K D = 3.16 and 3.54 ng/mL protein for MM-LDL and ox-LDL, respectively). (FITC) KP6 bound to lysophosphatidylcholine and oxidized phosphatidylcholine, both present abundantly in ox-LDL and MM-LDL, respectively. In vitro, (FITC)KP6 was detected on the surface and/or in the cytosol of human THP-1-derived macrophages incubated with ox-LDL and MM-LDL, but not LDL. These results suggest that (FITC)KP6 could be an efficient fluorescent probe for the specific detection of ox-LDL and MM-LDL and can therefore contribute to the identification, diagnosis, prevention, and treatment of atherosclerosis.

Angiotensin peptides attenuate platelet-activating factor-induced inflammatory activity in rats.

Angiotensin (Ang)--a peptide that is part of the renin-angiotensin system-induces vasoconstriction and a subsequent increase in blood pressure; Ang peptides, especially AngII, can also act as potent pro-inflammatory mediators. Platelet-activating factor (PAF) is a potent phospholipid mediator that is implicated in many inflammatory diseases. In this study, we investigated the effects of Ang peptides (AngII, AngIII, and AngIV) on PAF-induced inflammatory activity. In experiments using a rat hind-paw oedema model, AngII markedly and dose-dependently attenuated the paw oedema induced by PAF. The inhibitory effects of AngIII and AngIV on PAF-induced paw oedema were lower than that of AngII. Two Ang receptors, the AT1 and AT2 receptors, did not affect the AngII-mediated attenuation of PAF-induced paw oedema. Moreover, intrinsic tyrosine fluorescence studies demonstrated that AngII, AngIII, and AngIV interact with PAF, and that their affinities were closely correlated with their inhibitory effects on PAF-induced rat paw oedema. Also, AngII interacted with metabolite/precursor of PAF (lyso-PAF), and an oxidized phospholipid, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), which bears a marked structural resemblance to PAF. Furthermore, POVPC dose-dependently inhibited AngII-mediated attenuation of PAF-induced paw oedema. These results suggest that Ang peptides can attenuate PAF-induced inflammatory activity through binding to PAF and lyso-PAF in rats. Therefore, Ang peptides may be closely involved in the regulation of many inflammatory diseases caused by PAF.

A synthetic biotinylated peptide, BP21, inhibits the induction of mRNA expression of inflammatory substances by oxidized- and lyso-phosphatidylcholine.

Preclinical Research Oxidized low-density lipoprotein (ox-LDL) is implicated in many inflammatory diseases, e.g., type 2 diabetes, obesity, atherosclerosis, and metabolic syndrome. We previously reported that a synthetic biotinylated peptide, BP21, inhibits the bioactivity of ox-LDL via direct binding to ox-LDL. Here, we investigated the effect of BP21 on the mRNA expression of proinflammatory mediators induced by two major components of ox-LDL, oxidized- and lyso-phosphatidylcholine (ox-PC and LPC), in monocytes/macrophages (THP-1 cells) and adipocytes (3T3-L1 cells). In THP-1 cells, BP21 markedly reduced the mRNA expression of interleukin (IL)-6, adipocyte fatty acid-binding protein (aP2), tumor necrosis factor-α, and mitogen-activated protein kinase phosphatase-1, which are induced by one of the major bioactive components of ox-PC, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), and reduced the mRNA expression of IL-6, the ox-LDL-specific scavenger receptor CD36, and aP2 induced by LPC. In adipocytes, the mRNA expression of IL-1ß as an adipokine and aP2 is highly induced by ox-PC and LPC, and BP21 markedly reduced the mRNA expression of IL-1ß and aP2 induced by POVPC and LPC. Furthermore, BP21 specifically bound to LPC and POVPC in a dose-dependent manner. These results suggest that BP21 may be useful lead for the potential treatment and prevention of inflammatory diseases caused by ox-PC and LPC.

Common mechanism in endothelin-3 and PAF receptor function for anti-inflammatory responses.

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. Under inflammatory conditions, PAF is biosynthesized through the remodelling pathway and elicits many inflammatory responses through binding to its specific PAF receptor. Endogenous bioactive endothelins (ETs: ET-1, -2, and -3) are also considered potent inflammatory mediators that play a critical role in many inflammatory diseases. In this perspective, we provide a brief overview of possible common mechanisms in ETs and PAF receptor function for inflammatory responses. Accumulating evidence strongly suggests that ET-3, but not ET-1 and ET-2, can attenuate PAF-induced inflammation through direct binding of the Tyr-Lys-Asp (YKD) region in the peptide to PAF and its metabolite/precursor lyso-PAF, followed by inhibition of binding between PAF and its receptor. Additionally, YKD sequence-containing peptides may be useful as a novel type of anti-inflammatory drugs targeting this mechanism. These findings should lead to new treatment strategies for numerous inflammatory diseases by targeting the common mechanism in ET and PAF receptor function.

Synthetic biotinylated peptide compound, BP21, specifically recognizes lysophosphatidylcholine micelles.

Lysophosphatidylcholine, a major phospholipid component of oxidized low-density lipoprotein, is implicated in many inflammatory diseases, including atherosclerosis. We previously reported that Asp-hemolysin-related synthetic peptide (P21) composed of 21 amino acid residues markedly inhibits the bioactivities of oxidized low-density lipoprotein and lysophosphatidylcholine, by directly binding to oxidized low-density lipoprotein and lysophosphatidylcholine. Here, to clarify whether P21 specifically binds to lysophosphatidylcholine and what forms of lysophosphatidylcholine with which P21 interact, we investigated the interaction between P21 containing two tryptophan residues and lysophosphatidylcholine by using fluorescence spectroscopy, polyacrylamide gel electrophoresis, and surface plasmon resonance. From tryptophan fluorescence measurements, N-terminally biotinylated P21 specifically interacted with lysophosphatidylcholine, at concentrations exceeding the critical micelle concentration. From tryptophan fluorescence quenching, the tryptophan residues in biotinylated P21 in the presence of lysophosphatidylcholine were mostly exposed on the outer side of the peptide. From polyacrylamide gel electrophoresis and surface plasmon resonance, bound to 1-palmitoyl-lysophosphatidylcholine at concentrations higher than 100 µm, ensuring stable micelles. These results indicate that biotinylated P21 specifically recognizes lysophosphatidylcholine micelles. Further study of the interaction between biotinylated P21 and lysophosphatidylcholine micelles may provide important information for the prevention and treatment for many inflammatory diseases caused by lysophosphatidylcholine micelles.

Synthetic biotinylated peptide compounds derived from Asp-hemolysin: novel potent inhibitors of platelet-activating factor.

Platelet-activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent inflammatory mediator, is implicated in many inflammatory diseases and may possibly serve as a direct target for anti-inflammatory drugs. We have previously reported that Asp-hemolysin-related synthetic peptides (P4-P29) inhibit the bioactivities of oxidized low-density lipoprotein (ox-LDL) containing PAF-like lipids by direct binding to ox-LDL, which plays a key role in the atherosclerotic inflammatory process. In this study, we investigated whether these peptides inhibit the bioactivities of PAF by binding to PAF and its metabolite/precursor lyso-PAF. In in vitro experiments, P21, one of the peptides, bound to both PAF and lyso-PAF in a dose-dependent manner and markedly inhibited PAF-induced apoptosis in human umbilical vein endothelial cells. Moreover, in in vivo experiments, P4 and P21, particularly their N-terminally biotinylated peptide compounds (BP4 and BP21), inhibited PAF-induced rat paw oedema dose dependently and markedly, and showed sufficient inhibition of the oedema even at doses 150-300 times less than the doses of PAF antagonists. These results provide evidence that direct binding of N-terminally biotinylated peptide compounds derived from Asp-hemolysin to PAF and lyso-PAF leads to a dramatic inhibition of the bioactivities of PAF, both in vitro and in vivo, and strongly suggesting that these compounds may be useful as a novel type of anti-inflammatory drug for the treatment of several inflammatory diseases caused by PAF.

A synthetic peptide (P-21) derived from asp-hemolysin inhibits the induction of macrophage proliferation by oxidized low-density lipoprotein.

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (OxLDL) induces macrophage proliferation via the specific uptake of lysophosphatidylcholine (LysoPC) of OxLDL by class A, type I and type II macrophage scavenger receptors. We have previously shown that Asp-hemolysin from Aspergillus fumigatus binds to LysoPC as a typical lipid moiety of OxLDL. This study investigated the effect of the Asp-hemolysin-related peptide (P-21), a synthetic peptide derived from a region of Asp-hemolysin that is rich in positive charges, on macrophage proliferation induced by OxLDL. Mouse peritoneal macrophages were used for proliferation study. OxLDL induced macrophage proliferation in an oxidation time-dependent manner, and P-21 inhibited OxLDL-induced macrophage proliferation in a dose-dependent manner. Furthermore, the binding analysis of P-21 to OxLDL by dissociation-enhanced lanthanide fluorometric immunoassay indicated that P-21 binds to OxLDL. These results indicate that P-21 inhibits the OxLDL-induced macrophage proliferation through binding of P-21 to OxLDL.