Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.     

Peptides of major basic protein and eosinophil cationic protein activate human mast cells

The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca²⁺ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99–110 (MBP (99–110)) and ECP residues 29–45 (ECP (29–45)), respectively, induced degranulation of HCMCs and intracellular Ca²⁺ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99–110) or ECP (29–45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99–110) and ECP (29–45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.     

Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein

The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.     

Structural determinants of the eosinophil cationic protein antimicrobial activity

Abstract Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil.     

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP).

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN') and uridine and cytidine 2',3'-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the 'non-secretory' liver-type enzymes rather than to the 'secretory' pancreatic-type RNases.     

Topography studies on the membrane interaction mechanism of the eosinophil cationic protein

The eosinophil cationic protein (ECP) is an antipathogen protein involved in the host defense system. ECP displays bactericidal and membrane lytic capacities [Carreras et al. (2003) Biochemistry 42, 6636-6644]. We have now characterized in detail the protein-membrane interaction process. All observed fluorescent parameters of the wild type and single-tryptophan-containing mutants, as well as the results of decomposition analysis of protein fluorescence, suggest that W10 and W35 belong to two distinct spectral classes I and III, respectively. Tryptophan residues were classified and assigned to distinct structural classes using statistical approaches based on the analysis of tryptophan microenvironment structural properties. W10 belongs to class I and is buried in a relative nonpolar, nonflexible protein environment, while W35 (class III) is fully exposed to free water molecules. Tryptophan solvent exposure and the depth of the protein insertion in the lipid bilayer were monitored by the degree of protein fluorescence quenching by KI and brominated phospholipids, respectively. Results indicate that W35 partially inserts into the lipid bilayer, whereas W10 does not. Further analysis by electron microscopy and dynamic light scattering indicates that ECP can destabilize and trigger lipid vesicle aggregation at a nanomolar concentration range, corresponding to about 1:1000 protein/lipid ratio. No significant leakage of the vesicle aqueous content takes place below that protein concentration threshold. The data are consistent with a membrane destabilization "carpet-like" mechanism.     

A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein

Eosinophil cationic protein (ECP), a human RNAseA superfamily member, highly implicated in asthma pathology, is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In this study, we show that cell surface-bound heparan sulfate proteoglycans serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas-2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan-defective cells compared with their wild-type counterparts. Furthermore, pharmacological treatment combined with siRNA knockdown identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and ADP-ribosylating factor 6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and phosphatidylinositol-3-kinase activities, and is compatible with the characteristics of raft-dependent macropinocytosis. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.     

Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein

Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.     

Crystal structure of eosinophil cationic protein at 2.4 A resolution

Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.     

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 >/=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.     

Growth inhibition of mammalian cells by eosinophil cationic protein.

Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC(50) values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were estimated to be approximately 1-5 microm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.     

The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form

The human eosinophil cationic protein (ECP), also known as RNase 3, is an eosinophil secretion protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial lipopolysaccharides and heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for ligand binding studies. By exploring the protein-sulfate complex, we have defined the sulfate binding site architecture. Three main sites (S1-S3) are located in the protein active site; S1 and S2 overlap with the phosphate binding sites involved in RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of ligand anionic groups. The location of additional sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for glycosaminoglycans and can assist in structure-based drug design of inhibitors of the protein cytotoxicity to host tissues during inflammation.     

Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution

Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.     

Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants.Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38).Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity.The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides.