Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease.

Abstract

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) were isolated from lysates of human eosinophil granules by gel filtration and ion exchange chromatography on heparin-Sepharose. Radioimmunoassay, using monoclonal antibodies, of fractions from the heparinSepharose chromatography showed one peak of EDN activity and two peaks of ECP activity (termed ECP-1 and ECP-2).EDN, ECP-1, and ECP-2 each exhibited heterogeneity in charge and molecular weight when analyzed by two-dimensional nonequilibrium pH gradient electrophoresis and NaDodSO4/PAGE. Digestion of EDN with endoglycosidase F (endo F) decreased its molecular weight and charge heterogeneity. Thus, EDN likely contains a single complex oligosaccharide. Endo F digestion of ECP-1 and ECP-2 decreased the molecular weight of both polypeptides, indicating that both likely contain at least one complex oligosaccharide. Amino acid sequence analyses showed that ECP-1 and ECP-2 are identical from residue 1 through residue 59 and that the sequences of EDN and ECP are highly homologous (37 of 55 residues identical). Both EDN and ECP NH2-termnu*al sequences showed significant homology to RNase, especially in regions of the RNase molecule involved in ligand binding. EDN, ECP-1, and ECP-2 had neurotoxic activity, causing the Gordon phenomenon at doses down to 0.15 ,ug when injected into the cisterna magna; the proteins were comparable in their activities. These results indicate that EDN and ECP are related proteins and suggest that they derived from genes associated with the RNase family.The human eosinophil granule contains several cationic proteins including the major basic protein (1-3), the eosinophil cationic protein (ECP) (4,5), the eosinophilderived neurotoxin (EDN) (6, 7), and the eosinophil peroxidase (EPO) (8). Major basic protein and ECP are potent helminthotoxins (9-11), and major basic protein is toxic to mammalian cells (9, 12). When injected intrathecally into rabbits or guinea pigs, both EDN and ECP, but not major basic protein or EPO, produce the Gordon phenomenon, a neurologic syndrome characterized in the rabbit by stiffness, ataxia, muscle weakness, and muscle wasting (6,7,13 MATERIALS AND METHODSEosinophil Granules. From nine patients with marked peripheral blood eosinophilia, eosinophils were collected by cytapheresis with hydroxyethyl-starch (15). The mean percentage of eosinophils in the concentrate was 79% (range, 64-94%); between 2 x 1010 and 2 x 1011 eosinophils were collected from a single patient. For purification of the eosinophil granules from the large numbers of cells obtained, we used procedures described previously (1-3, 7, 16). The enriched granule fractions were transferred to 2-ml freezing vials (Nunc) and stored in liquid nitrogen for up to 3 years.EDN and ECP Isolation. Frozen granule pellets were thawed, adjusted to pH 3 with 0.1 M HCl, and centrifuged at 40,000 x g for 20 min. Supernatants from the granule extracts were applied to a 1.2 x 48 cm Sephadex G-50 column equilibrated with 0.025 M NaOAc/0.15 M Na...

Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.

“…Fig. 2 b shows the alignment of the cDNA-derived amino acid sequence of ECP (beginning at the arginine at position 1 of the NH2 terminus of the granuleextracted protein [3]) with the sequences of EDN (19) (shown to be identical to HNSR [19,20]), human liver ribonuclease (HLR) (32), HPR (33), and angiogenin (ANG) (34,35). The sequences of EDN, HNSR, and the 25 NH2-terminal residues of HLR are identical.…”

New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us

“…Its amino acid sequence, deduced from the DNA sequence (Hamann et al, 1990), is identical to the chemically determined sequence of RNase Us found in human urine (Beintema et al, 1988), except for the amino acid residue at position 7. Whereas a tryptophan residue was predicted from the DNA sequence of EDN, this amino acid could not be identified by chemical means in RNase Us (Beintema et al, 1988) or in EDN (Gleich et al, 1986). This suggests a posttranslational modification of this residue.…”

“…For instance, eosinophil derived neurotoxin (EDN)' was originally identified by its neurotoxic effect after injection into the cerebellum (Durack et al, 1981). This protein was subsequently shown to contain RNase activity and to be structurally homologous to bovine pancreatic RNase A (Gleich et al, 1986;Slifman et al, 1986). Its amino acid sequence, deduced from the DNA sequence (Hamann et al, 1990), is identical to the chemically determined sequence of RNase Us found in human urine (Beintema et al, 1988), except for the amino acid residue at position 7.…”

Reactivity of monoclonal antibodies EG1 and EG2 with eosinophils and their granule proteins

“…EG1 was derived from a mouse injected with extracts of unstimulated eosinophils, and EG2 was derived from a mouse injected with secreted proteins from zymosan-C3b-stimulated eosinophils [12]. EG1 was thought to recognize both stored and secreted forms of eosinophil cationic protein (ECP), whereas EG2 was thought to recognize only secreted ECP and eosinophil-derived neurotoxin (EDN) [12]; amino acid sequencing and cloning of ECP and EDN [13][14][15][16][17] showed that these molecules are homologous and are members of the RNase superfamily. This report [12] further suggested that EG2 could be used to identify activated eosinophils in tissues, and a later report [18] confirmed the apparent ability of EG2 to identify activated eosinophils.…”