Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease.
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 heparin-Sepharose 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, END 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-terminal 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 micrograms 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.
Gleich
G J
GJ
Loegering
D A
DA
Bell
M P
MP
Checkel
J L
JL
Ackerman
S J
SJ
McKean
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D001798:Blood Proteins / Q000302:isolation & purification* / Q000633:toxicity; D003594:Cytoplasmic Granules / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D009498:Neurotoxins / Q000302:isolation & purification* / Q000633:toxicity; D011817:Rabbits; D012260:Ribonucleases
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D006023:Glycoproteins; D009498:Neurotoxins; D012260:Ribonucleases; D006026:Glycoside Hydrolases