Amino-terminal processing of chemokine ENA-78 regulates biological activity.
Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a potent stimulator of neutrophils, inducing a variety of biological responses such as chemotaxis, enzyme release, up-regulation of surface receptors, and intracellular calcium mobilization. Proteolysis of ENA-78 with cathepsin G and chymotrypsin yielded a time-dependent increase in elastase-releasing activity, predicting the formation of truncation products with higher potency than native ENA-78. To investigate the biological implications of progressive truncation of ENA-78, the N-terminal variants ENA(5-78), ENA(9-78), and ENA(10-78) were cloned and expressed in E. coli. When tested in the neutrophil elastase release assay, the variants ENA(5-78) and ENA(9-78) had a 2-3-fold higher potency than full-length ENA-78, while ENA(10-78) was 3-fold less potent. In the chemotaxis assay, the variant ENA(5-78) exhibited an 8-fold and ENA(9-78) a 2-fold higher potency than native ENA-78. ENA(10-78), conversely, was 10-fold less potent, but reached a comparable efficacy to ENA-78 at 10(-)7 M concentration. In summary, the rank order in potency with respect to elastase release was ENA(9-78) > ENA(5-78) > ENA-78 > ENA(10-78), while for chemotaxis it was ENA(5-78) > ENA(9-78) > ENA-78 > ENA(10-78). Variant ENA(5-78) had a higher overall potency and efficiency for chemotaxis than interleukin-8 (IL-8), while ENA(9-78) exhibited a higher efficiency at concentrations of 1-100 nM. The fact that neutrophil cathepsin G produces the stable ENA(9-78) variant in vitro strongly suggests a role for this N-terminal proteolysis during inflammatory processes in vivo.
Nufer
O
O
Theodor Kocher Institute, University of Bern, Switzerland.
Corbett
M
M
Walz
A
A
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D054365:Chemokine CXCL5; D019743:Chemokines, CXC / Q000097:blood / Q000235:genetics / Q000502:physiology; D002634:Chemotaxis, Leukocyte; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016209:Interleukin-8 / Q000031:analogs & derivatives* / Q000097:blood / Q000235:genetics / Q000502:physiology; D019272:Leukocyte Elastase / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D018375:Neutrophil Activation; D009504:Neutrophils / Q000201:enzymology / Q000502:physiology; D010446:Peptide Fragments / Q000097:blood* / Q000235:genetics / Q000502:physiology; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
C486263:CXCL5 protein, human; D054365:Chemokine CXCL5; D019743:Chemokines, CXC; D016209:Interleukin-8; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D019272:Leukocyte Elastase; D002118:Calcium