Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences.
Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. The key membrane-associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22-phox and gp91-phox. Currently, only the human, porcine, murine, and rattus p22-phox and the human, porcine, and murine gp91-phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22-phox and gp91-phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91-phox and p22-phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis.
Department of Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA.
A R
AR
Davis
P L
PL
Mascolo
P L
PL
Bunger
K M
KM
Sipes
M T
MT
Quinn
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase