Abstract
A novel eosinophil chemotactic cytokine (ECF-L) was purified from the culture supernatant of splenocytes of mice by a combination of anion-exchange chromatography, Procion red-agarose affinity chromatography, size exclusion high performance liquid chromatography (HPLC), and reverse phase HPLC. The NH(2)-terminal amino acid sequence was determined by direct protein sequencing. An ECF-L cDNA clone of 1,506 nucleotides was isolated from a cDNA library, and the nucleotide sequence predicted a mature protein of 397 amino acids. A recombinant ECF-L showed a level of eosinophil chemotactic activity comparable with that of natural ECF-L, and the activity was inhibited by a monoclonal antibody to ECF-L. ECF-L also attracted T lymphocytes and bone marrow polymorphonuclear leukocytes in vitro, whereas it caused selective extravasation of eosinophils in vivo. ECF-L mRNA was highly expressed in spleen, bone marrow, lung, and heart. A comprehensive GenBank data base search revealed that ECF-L is a chitinase family protein. ECF-L retains those amino acids highly conserved among chitinase family proteins, but Asp and Glu residues essential for the proton donation in hydrolysis were replaced by Asn and Gln, respectively. Although ECF-L contains a consensus CXC sequence near the NH(2) terminus akin to chemokine family proteins, the rest of ECF-L shows poor homology with chemokines.
Highlights
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number D87757
Previous studies have shown that a splenocyte-derived eosinophil chemotactic factors (ECFs) (ECF-L) is produced in an antigen-specific manner during the course of parasitic infections accompanying systemic and local eosinophilia [7], and CD4ϪCD8ϩ T cells play an important role in ECF-L production [10]
The results clearly show that ECF-L is a novel eosinophil chemotactic cytokine that belongs to the chitinase protein family
Summary
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number D87757. The results clearly show that ECF-L is a novel eosinophil chemotactic cytokine that belongs to the chitinase protein family. Purification of ECF-L—For purification of ECF-L protein, the medium conditioned by spleen cells obtained from S. japonicum-infected mice was concentrated, dialyzed against Tris-HCl buffer, and applied to a DE52 anion-exchange column.
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