Effect of the R569W missense mutation on the biosynthesis of myeloperoxidase.
Human neutrophil microbicidal activity is largely mediated by reactive species generated by the oxygen-dependent myeloperoxidase (MPO) system. Peroxidase-negative neutrophils from many patients with hereditary MPO deficiency possess a 90-kDa MPO-related protein. We recently identified a missense mutation, R569W, in the MPO gene of many subjects with MPO deficiency. In these studies we examined the consequences of R569W on MPO biosynthesis and processing, using stably transfected K562 cells expressing normal MPO or the R569W mutation. K562 cells expressing normal MPO mimicked faithfully many features of MPO biosynthesis in myeloid cells. 1) apopro-MPO was synthesized; 2) a functional heme group was inserted into apopro-MPO, and enzymatically active pro-MPO was thereby generated; 3) pro-MPO underwent proteolytic processing to mature MPO; and 4) hemin augmented the processing of pro-MPO. pREP-R569W cells synthesized apopro-MPO, but heme was not inserted. Neither enzymatically active pro-MPO nor mature MPO was synthesized by transfectants expressing mutated cDNA, confirming our hypothesis that the R569W mutation results in a form of apopro-MPO which does not undergo post-translational processing to enzymatically active MPO species. In addition, these data support previous suggestions that heme insertion into apopro-MPO is necessary for its subsequent proteolytic processing into mature MPO subunits.
Nauseef
W M
WM
Department of Medicine, Veterans Administration Medical Center, Iowa City, Iowa 52242, USA.
Cogley
M
M
McCormick
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001770:Blood Bactericidal Activity; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D006418:Heme / Q000494:pharmacology; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000201:enzymology* / Q000502:physiology; D009195:Peroxidase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006418:Heme; D009195:Peroxidase