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Published on: March 15, 2016
Biological activity of the growth factor-induced cytokine N51: structure-function analysis using N51/Interleukin-8
J N Heinrich1, E C O'Rourke, L Chen
1Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Insights
The N51/KC protein, distinct from interleukin-8 (IL-8), induces chemotaxis and calcium flux in neutrophils via unique receptor interactions. Structural analysis reveals key regions for receptor binding and biological activity in these chemokines.
Area of Science:
- Molecular and Cellular Biology
- Immunology
- Biochemistry
Background:
- Immediate-early gene N51/KC encodes a protein with chemokine-like functions.
- Interleukin-8 (IL-8) is a well-characterized chemokine involved in neutrophil recruitment.
Purpose of the Study:
- To characterize the biological activities and receptor interactions of the N51/KC protein.
- To compare the functional properties of N51/KC with those of IL-8.
- To identify structural determinants of N51/KC and IL-8 responsible for receptor specificity and biological activity.
Main Methods:
- Expression and purification of N51/KC protein using the baculovirus system.
- Chemotaxis and intracellular Ca2+ flux assays in human neutrophils.
- Radioligand binding assays using 125I-labeled N51/KC and IL-8.
- Competition binding studies with N51/KC and IL-8.
- Analysis of N51/KC and IL-8 mutants and hybrids.
Main Results:
- N51/KC induces chemotaxis and intracellular Ca2+ flux in human neutrophils.
- N51/KC binds to human neutrophils with lower affinity than IL-8 and interacts with distinct receptor populations.
- Specific regions within N51/KC and IL-8, particularly between cysteines 2 and 3, are critical for receptor recognition.
- The C-terminus is essential for the biological activity of both N51/KC and IL-8 but does not determine receptor specificity.
Conclusions:
- N51/KC represents a distinct chemokine with unique receptor binding characteristics compared to IL-8.
- Structural analysis of N51/KC and IL-8 hybrids provides insights into chemokine-receptor interactions.
- The findings contribute to understanding the molecular basis of chemokine signaling and neutrophil activation.
Abstract:
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.
