Biological activity of the growth factor-induced cytokine N51: structure-function analysis using N51/Interleukin-8 chimeric molecules.
The immediate-early gene N51/KC encodes a protein which following expression in the baculovirus system and purification to apparent homogeneity is able to induce chemotaxis and intracellular Ca2+ flux, to compete for 125I-labeled interleukin-8 (IL-8) binding, and upon iodination, to bind specifically to human neutrophils. The activity of N51/KC can be distinguished from that of IL-8 by a number of criteria. First, at equivalent concentrations, the specific binding of [125I]N51/KC to human neutrophils is about 10 times less than that of [125I]IL-8. Second, the competition studies of [125I]IL-8 with IL-8 define a single class of high-affinity receptors, while the presence of both a high- and a low-affinity class of receptors is defined by N51/KC. Third, although the changes in intracellular Ca2+ of fura-2/AM-preloaded human neutrophils elicited by N51/KC and IL-8 are similar, pretreatment of the cells with N51/KC did not result in a loss of response to a subsequent treatment with IL-8; in contrast, treatment with IL-8 did result in the subsequent desensitization to N51/KC. To further characterize N51/KC, mutants and hybrids of N51/KC and IL-8 were produced and analyzed for the ability to compete for [125I]IL-8 binding and elicit intracellular Ca2+ changes in human neutrophils. Two important observations came from these studies. First, the N51/IL-8I hybrid in which the N51/KC sequence between cysteines 2 and 3 (or first disulfide bond) is replaced by the corresponding sequence in IL-8 shows IL-8-like properties, indicating that this region is important for specific receptor recognition. Second, the N51 delta III and IL-8 delta III C-terminus deletion mutants were biologically inactive, but the hybrid molecules N51/IL-8III and IL-8/N51III, in which the C termini were exchanged, had biological activities similar to that of the wild-type molecules, demonstrating that the presence of the C terminus is essential for the biological activity of these chemokines but does not confer receptor specificity.
Heinrich
J N
JN
Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
O'Rourke
E C
EC
Chen
L
L
Gray
H
H
Dorfman
K S
KS
Bravo
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001667:Binding, Competitive; D002118:Calcium / Q000097:blood; D002460:Cell Line; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D015870:Gene Expression; D017781:Genes, Immediate-Early; D006133:Growth Substances / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D009504:Neutrophils / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D018123:Receptors, Interleukin / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D009838:Oligodeoxyribonucleotides; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D002118:Calcium