Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.
Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/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 alpha/beta 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 (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity 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.
Jerva
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Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Sullivan
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Lolis
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D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D054426:Chemokine CXCL2; D019743:Chemokines, CXC; D003001:Cloning, Molecular; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D015846:Monokines / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000378:metabolism; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D015703:Antigens, CD; C499706:CXCL2 protein, human; D054426:Chemokine CXCL2; D019743:Chemokines, CXC; D017931:DNA Primers; D000939:Epitopes; D036341:Intercellular Signaling Peptides and Proteins; D015846:Monokines; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins