Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs

Neutrophils play an essential role in bovine cellular host defense, and com promised leukocyte function has been linked to the development of respirato ry and mucosal infections, During the host defense process, neutrophils mig rate into infected tissues where they become activated, resulting in the as sembly of neutrophil membrane and cytosolic proteins to form a superoxide a nion-generating complex known as the NADPH oxidase, Two of the essential cy tosolic components of the NADPH oxidase are p47-phox and p67-phox, Currentl y, only the human and murine homologs of these proteins have been sequenced . Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and express bovine p47-phox and p6 7-phox, Using polymerase chain reaction (PCR) cloning techniques and a bovi ne bone marrow cDNA library, we have cloned both of these bovine NADPH oxid ase cytosolic components. Comparison of the bovine sequences with those of the human and murine homologs showed that they were highly conserved, but a lso revealed important information regarding key structural features of p47 -phox and p67-phox, including location of putative phosphorylation sites. F unctional expression of bovine p47-phox and p67-phox showed that these prot eins could substitute for the human proteins in reconstituting NADPH oxidas e activity in a cell-free assay system, again demonstrating the high degree of conservation between human and bovine homologs, This study greatly cont ributes to our understanding of the potential structural/functional regions of p47-phox and p67-phox as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases.