Human eosinophil cytotoxicity-enhancing factor. Eosinophil-stimulating and dithiol reductase activities of biosynthetic (recombinant) species with COOH-terminal deletions.
U937 cells produce eosinophil cytotoxicity-enhancing factor (ECEF) polypeptides of 14 and 10 kDa that have identical NH2-terminal amino acid sequences. The 10-kDa form has greater eosinophil-stimulating activity (half-maximal at > 20-fold lower concentration). We considered the hypothesis that there is a precursor-product relationship between the 14- and 10-kDa species. Recombinant 14-kDa 104-amino acid ECEF (rECEF-104) had a slight stimulatory effect on eosinophil cytotoxic function at concentrations of 160 nM and above. In contrast, two species, rECEF-80 and rECEF-84, representing cleavage products of approximately 10 kDa had substantial statistically significant cytotoxicity-enhancing activity at concentrations as low as 10 pM. This evidence demonstrates the potential to generate the high-activity ECEF species by proteolytic cleavage of the 104-amino acid species. Another feature of this cytokine is the sequence from amino acids 31 to 34, which constitutes the conserved and active site of the enzyme thioredoxin. When tested for dithiol reductase enzymatic activity, rECEF-104 was active in a dose- and time-dependent manner, whereas the truncated forms of the molecule had no dithiol reductase activity. Thus the eosinophil-stimulating functions of the molecule do not correlate with its enzymatic activity. The evidence shows that the enzymatic activity is not essential for the initial interaction of ECEF with the eosinophil, and it suggests that the ECEF molecule functions by means of two discrete mechanisms.
Silberstein
D S
DS
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
McDonough
S
S
Minkoff
M S
MS
Balcewicz-Sablinska
M K
MK
D001483:Base Sequence; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D004804:Eosinophils / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase