Cell Penetrating Peptide Derived from Human Eosinophil Cationic Protein Decreases Airway Allergic Inflammation.

Cell penetrating peptide derived from human eosinophil cationic protein (CPPecp) is a 10-amino-acid peptide containing a core heparan sulfate (HS)-binding motif of human eosinophil cationic protein (ECP). It binds and penetrates bronchial epithelial cells without cytotoxic effects. Here we investigated airway-protective effects of CPPecp in BEAS-2B cell line and mite-induced airway allergic inflammation in BALB/c mice. In BEAS-2B cell, CPPecp decreases ECP-induced eotaxin mRNA expression. CPPecp also decreases eotaxin secretion and p-STAT6 activation induced by ECP, as well as by IL-4. In vivo studies showed CPPecp decreased mite-induced airway inflammation in terms of eosinophil and neutrophil count in broncho-alveolar lavage fluid, peri-bronchiolar and alveolar pathology scores, cytokine production in lung protein extract including interleukin (IL)-5, IL-13, IL-17A/F, eotaxin; and pause enhancement from methacholine stimulation. CPPecp treated groups also showed lower serum mite-specific IgE level. In this study, we have demonstrated the in vitro and in vivo anti-asthma effects of CPPecp.

Low-molecular-weight heparin and unfractionated heparin decrease Th-1, 2, and 17 expressions.

BACKGROUND: We evaluated the effects of T helper cell differentiation in a mite-allergic animal model treated with inhaled heparins of different molecular weight. METHOD: BALB/c mice were divided into four groups: 1. Control, 2. Mite intratracheal (mIT), 3. Inhaled heparin (hIN), 4. Inhaled low-molecular-weight heparin (lmwhIN). Groups 2, 3, and 4 were sensitized twice with Der p allergen subcutaneously on day 1 and day 8. Der p allergen was administered intratracheally on day 15. Groups 3 and 4 were treated with heparin or low-molecular-weight (lmw) heparin intranasally from day 1 to 22. Splenocytes from sacrificed mice stimulated with 16 µg/ml of Der p were cultured for 72 hours. Supernatants of splenocyte were collected to analyze the effect of Interleukin (IL)17-A/F, Interferon(IFN)-γ, IL-4, IL-13, and IL-10. Serum was also collected for Der P-specific IgE level on day 23. Total RNA was extracted from spleen tissue for mRNA expression. Gene expression of Foxp3, IL-10 IFN-γ, GATA3, IL-5, and RORγt were analyzed. RESULTS: Both hIN and lmwhIN groups had lower serum IgE level than that of the mIT group (both p<0.0001). Both hIN and lmwhIN groups showed significantly decreased transcripts of GATA-3, IFN-γ, IL-5, and RORγt mRNA in their spleen. Regarding the supernatant of splenocyte culture stimulated with Der p, compared with the mIT group, there were significant decreases in IL-17A/F, IFN-γ, IL-4, IL-13, and IL-10 secretion in inhaled hIN and lmwhIN groups. CONCLUSIONS: From this balb/c mice study, the analyses of mRNA and cytokines revealed that both intranasal heparin and lmw heparin treatment decreased the expression of Th1, Th2, and Th17 in spleen. The underlying mechanism(s) warrant further studies.

In silico prediction and in vitro characterization of multifunctional human RNase3.

Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, (34)RWRCK(38) (HBR1), (75)RSRFR(79) (HBR2), and (101)RPGRR(105) (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members.

A novel cell-penetrating peptide derived from human eosinophil cationic protein.

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.

Synthesis of heparin oligosaccharides and their interaction with eosinophil-derived neurotoxin.

A convenient route for the synthesis of heparin oligosaccharides involving regioselective protection of D-glucosamine and a concise preparation of rare L-ido sugars from diacetone α-D-glucose is described. Stereoselective coupling of a D-glucosamine-derived trichloroacetimidate with a 1,6-anhydro-ß-L-idopyranosyl 4-alcohol gave the desired α-linked disaccharide, which was used as repeating unit for dual chain elongation and termination. Stepwise assembly from the reducing to the non-reducing end with a D-glucosamine-derived monosaccharide as starting unit furnished the oligosaccharide skeletons having different chain lengths. A series of functional group transformations afforded the expected heparin oligosaccharides with 3, 5 and 7 sugar units. Interaction of these oligosaccharides with eosinophil-derived neurotoxin (EDN), a cationic ribonuclease and a mediator produced by human eosinophils, was further investigated. The results revealed that at 5 µg mL(-1), the heptasaccharide has sufficiently strong interference to block EDN binding to Beas-2B cells. The tri- and pentasaccharides have moderate inhibitory properties at 50 µg mL(-1) concentration, but no inhibition has been observed at 10 µg mL(-1). The IC(50) values of the tri-, penta- and heptasaccharides are 69.4, 47.2 and 0.225 µg mL(-1), respectively.

TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

BACKGROUND: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis. RESULTS: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody. CONCLUSION: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

Characterization of molecular interactions between eosinophil cationic protein and heparin.

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.