Purification, sequence analysis, and biological characterization of a second bovine monocyte chemotactic protein-1 (Bo MCP-1B).
Madin Darby bovine kidney (MDBK) cells were used as a source to identify novel bovine chemotactic factors for granulocytes and monocytes. A major bovine granulocyte chemotactic protein (GCP-2) has previously been isolated. A novel bovine monocyte chemotactic protein (bo MCP) was produced on MDBK cells stimulated with phorbol ester. The 14-kDa protein was purified to homogeneity by adsorption to controlled pore glass, heparin affinity chromatography, cation-exchange FPLC, and RP-HPLC. The amino acid sequence of the NH2-terminally blocked protein was determined by Edman degradation using proteolytic fragments. The primary structure of the bo MCP, characterized by four conserved cysteines, allowed classification of the protein within the C-C chemokine family. Bo MCP-1B was most related to known human and bovine MCPs. Compared to bovine MCP-1 and MCP-2, the protein consists of 84% and 53% identical amino acids, respectively. Since this bo MCP was also most homologous to human and animal MCP-1, it was designated bo MCP-1B. The minimal effective dose of bo MCP-1B for monocyte chemotactic activity was 0.2 mM. The maximal migration index, reached at 2 nM, was comparable to that of natural human MCP-1. Furthermore, bo MCP-1B was found to be capable of stimulating beta-glucuronidase release from monocytes. In contrast, bo MCP-1B was not chemotactic for neutrophilic and eosinophilic granulocytes. By its biological and biochemical characteristics, bo MCP-1B has to be considered as an authentic additional MCP-1 chemokine. The existence of a possible human counterpart for this novel MCP-1B still needs to be elucidated.
Proost
P
P
Rega Institute, Laboratory of Molecular Immunology, University of Leuven, Belgium.
Wuyts
A
A
Lenaerts
J P
JP
Van Damme
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D005966:Glucuronidase / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007962:Leukocytes / Q000187:drug effects; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018932:Chemokine CCL2; D002630:Chemotactic Factors; D005966:Glucuronidase; D013755:Tetradecanoylphorbol Acetate