Biological activity of the growth factor-induced cytokine N51: structure-function analysis using N51/Interleukin-8 chimeric molecules.

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 -8-like properties, indicating that this region is important for specific receptor recognition. Second, the N51AIII and IL-8AIII C-terminus deletion mutants were biologically inactive, but the hybrid molecules N51/IL-8III and IL-8/N51111, 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.

N51 Competes 125I-Interleukin (IL)-8 Binding to IL-8Rβ but Not IL-8Rα

“…The results presented here are consistent with and extend our previous observations using human neutrophils (1). First, we have demonstrated that N51 can only displace I-IL-8 and confirms our speculation that the high affinity receptor for N51 in human neutrophils is the IL-8R␤ (1, 2).…”

“…: 609-252-5744; Fax: 609-252-6051. 1 The abbreviations used are: IL, interleukin; MGSA, melanoma growth-stimulating activity; MIP, macrophage inflammatory protein; MCP, monocyte chemotactic protein; NAP, neutrophil-activating peptide; PCR, polymerase chain reaction; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; GRO, growth-related gene; ENA, epithelial cell-derived neutrophil-activiting protein; ␥-IP, interferoninducible protein; RANTES, regulated on activation normal T-cell expressed and secreted.…”

“…The first two cysteines are either separated by one amino acid or adjacent to each other, and this distinction, which is correlated with functional properties (see below), is used to designate the members into two classes, the "C-X-C" or ␣-family and the "C-C" or ␤-family, respectively. For members whose three-dimensional structure has been determined, namely platelet factor-4, interleukin-8 (IL-8), 1 GRO␣ or melanoma growth-stimulating activity (GRO␣/MGSA), and macrophage inflammatory protein-1␤ (MIP-1␤), the cysteine motif appears to restrict the secondary structure to forming two disulfide bonds, one between the first and third cysteines and another between the second and fourth cysteines, resulting in similar tertiary structures. It is believed that all the members share this tertiary configuration, since the disulfide bonds are essential for the biological activity of chemokines for which this property has been tested (for reviews see Refs.…”

“…IL-8 (R & D Systems) was iodinated by the Bolton-Hunter method to a specific activity of 97.4 mCi/g (DuPont NEN Custom Iodination Laboratory) as described (1). The competition curve was performed with a 0.1-200-fold excess of competitor as compared with 3 nM 125 I-IL-8.…”

Neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes.

“…KC (also designated N51 in the murine system and termed N51/KC in this manuscript) is a rodent ␣ -chemokine related to the human chemokine interleukin-8 (IL-8) (20)(21)(22)(23)(24)(25)(26). N51/KC stimulates chemotaxis specifically of neutrophils, which exit rapidly from the circulation to provide the first line of cellular defense against invading pathogens.…”