Identification of a splice variant mRNA of p40phox, an NADPH oxidase component of phagocytes

Northern blot analysis using p40phox cDNA probe revealed that two sizes of p40phox mRNAs were expressed in human promyelocytic HL-60 and bone marrow c ells. To characterize these mRNAs, we performed reverse transcription using total RNA from HL-60 cells, and amplified the coding region of p40phox by polymerase chain reaction with oligonucleotide primers. Two cDNA fragments with different sizes were isolated, One was identical to a known p40phox cD NA (1054 bp) which encoded a protein of 339 residues (39031 Da) with a calc ulated pi of 6.5, The other cDNA (1299 bp) contained an additional 245 bp i ntron 8 sequence in the open reading frame and encoded a protein of 348 res idues (39000 Da) with a calculated pI of 9.3, N-terminal 253 residues mere identical between p40phox and the variant protein, whereas C-terminal 254-3 48 residues of the variant protein shared low homology with p40phox. Intere stingly, the variant protein lacked PC (Phox and Cdc24p) motif of p40phox, which is assumed to be important for the interaction with p67phox, In addit ion, Western blot analysis revealed that the variant protein,vas not detect ed in HL-60 cells and neutrophils, Together, these observations suggest tha t alternatively spliced variant mRNA of p40phox is expressed, but its prote in is hardly present in myeloid cells, (C) 1999 Federation of European Bioc hemical Societies.