6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking during pregnancy.
Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme's intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.
Kindzelskii
Andrei L
AL
Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, MI 48105, USA.
Ueki
Tatsuya
T
Michibata
Hitoshi
H
Chaiworapongsa
Tinnakorn
T
Romero
Roberto
R
Petty
Howard R
HR
D002469:Cell Separation; D003078:Colchicine / Q000494:pharmacology; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D005954:Glucosephosphate Dehydrogenase / Q000097:blood* / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D015591:Microspectrophotometry; D022101:Microtubule-Organizing Center / Q000201:enzymology / Q000378:metabolism; D009249:NADP / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D018375:Neutrophil Activation / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D016877:Oxidants / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000097:blood; D010734:Phosphogluconate Dehydrogenase / Q000097:blood* / Q000737:chemistry*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000097:blood* / Q000737:chemistry* / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000276:immunology; D026361:Reactive Nitrogen Species / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000097:blood
D046911:Macromolecular Substances; D016877:Oxidants; D011257:Pregnancy Proteins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009249:NADP; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D003078:Colchicine