Investigation of the contribution of histidine 119 to the conduction of protons through human Nox2.
The conduction of protons through human Nox2 has previously been shown to be dependent upon His115. Alignment of sequences for both animal and plant Nox proteins indicated that histidines 115 and 119 are both highly conserved, while His111 was conserved among animal homologues of Nox1-4. To investigate the possible role that these histidine residues might play in the conduction of protons through Nox2, we have introduced both paired and single mutations into these histidine residues. Each construct was used to generate a CHO cell line in which the expression of the mutated Nox2 was assessed. Nox2 was expressed in each of the CHO cell lines generated, however, the level of expression of H111/115L in CHO cells was lower and that of H111L very much reduced, compared to that of wild-type Nox2. The arachidonic acid activated proton flux was absent in the CHO cell lines expressing the mutations of H111/115L, H111/119L or H115/119L, compared to that observed for wild-type Nox2. Similarly only a small efflux of protons was observed from CHO cells expressing either H119L or H111L. In all cases the expected proton flux was elicited through the addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone. Conclusions regarding the role of His111 in the conduction of protons cannot be drawn due to the reduced expression. We can, however, conclude that His119, in addition to His115, is required for the conduction of protons through Nox2. His119 has been identified as a highly conserved residue for which no function has previously been proposed.
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.
Tosti J
TJ
Mankelow
X Wen
XW
Hu
Kate
K
Adams
Lydia M
LM
Henderson
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017494:Proton Pumps / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D017494:Proton Pumps; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D006639:Histidine; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases