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Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: Sequence analysis and mutagenesis identify receptor binding epitopes
Author(s) -
Jerva L. Fred,
Lolis Elias,
Sullivan Gail
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060805
Subject(s) - recombinant dna , mutagenesis , epitope , receptor , microbiology and biotechnology , epitope mapping , biology , chemistry , computational biology , genetics , mutation , antibody , gene
Murine macrophage inflammatory protein‐2 (MIP‐2), a member of the α‐ chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the α‐chemokines, such as interleukin‐8, gro‐α/MGSA, and neutrophil activating peptide‐2 (NAP‐2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP‐2 from Pichia pastoris . Circular dichroism spectroscopy reveals that MIP‐2 exhibits a highly ordered secondary structure consistent with the α/β structures of other chemokines. Recombinant MIP‐2 is chemotactic for human and murine neutrophils and up‐regulates cell surface expression of Mac‐1. MIP‐2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP‐2 to the human types A and B IL‐8 receptors and the murine homologue of the IL‐8 receptor. MIP‐2 displays low‐affinity binding to the type A IL‐8 receptor (K d >120 nM) and high‐affinity binding to the type B IL‐8 receptor (K d 5.7 nM) and the murine receptor (K d 6.8 nM). The three‐dimensional structure of IL‐8 and sequence analysis of six chemokines (IL‐8, gro‐α, NAP‐2, ENA‐78, KC, and MIP‐2) that display highaffinity binding to the IL‐8 type B receptor are used to identify an extended N‐terminal surface that interacts with this receptor. Two mutants of MIP‐2 establish that this region is also involved in binding and activating the murine homologue of the IL‐8 receptor. Differences in the sequence between IL‐8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL‐8 for binding to the type A receptor.