PURIFICATION, SEQUENCE-ANALYSIS, AND BIOLOGICAL CHARACTERIZATION OF A 2ND 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 controled 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 nM. 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.