PURIFICATION, SEQUENCE-ANALYSIS, AND BIOLOGICAL CHARACTERIZATION OF A2ND BOVINE MONOCYTE CHEMOTACTIC PROTEIN-1 (BO-MCP-1B)

Madin Darby bovine kidney (MDBK) cells were used as a source to identi fy novel bovine chemotactic factors for granulocytes and monocytes. A major bovine granulocyte chemotactic protein (GCP-2) has previously be en isolated. A novel bovine monocyte chemotactic protein (bo MCP) was produced on MDBK cells stimulated with phorbol ester. The 14-kDa prote in was purified to homogeneity by adsorption to controled pore glass, heparin affinity chromatography, cation-exchange FPLC, and RP-HPLC. Th e amino acid sequence of the NH2-terminally blocked protein was determ ined by Edman degradation using proteolytic fragments. The primary str ucture of the bo MCP, characterized by four conserved cysteines, allow ed classification of the protein within the C-C chemokine family. Bo M CP-1B was most related to known human and bovine MCPs. Compared to bov ine MCP-1 and MCP-2, the protein consists of 84% and 53% identical ami no acids, respectively. Since this bo MCP was also most homologous to human and animal MCP-1, it was designated bo MCP-1B. The minimal effec tive dose of bo MCP-1B for monocyte chemotactic activity was 0.2 nM. T he 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 o f stimulating beta-glucuronidase release from monocytes. In contrast, bo MCP-1B was not chemotactic for neutrophilic and eosinophilic granul ocytes. By its biological and biochemical characteristics, bo MCP-1B h as to be considered as an authentic additional MCP-1 chemokine. The ex istence of a possible human counterpart for this novel MCP-1B still ne eds to be elucidated.