Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells

Publisher Website
Google Scholar
Abstract
Myeloperoxidase is a major protein component of the azurophilic granules (specialized lysosomes) of normal human neutrophils and serves as part of a potent bactericidal system in the host defense function of these cells. In normal, mature cells, myeloperoxidase occurs exclusively as a dimer of Mr 150,000 while in immature leukemia cells, there are both monomeric (Mr 80,000) as well as dimeric species. Like other lysosomal enzymes, myeloperoxidase is synthesized as a larger glycosylated precursor (Mr 91,000) that undergoes processing through single-chain intermediates (Mr 81,000 and 74,000) to yield mature heavy (Mr 60,000) and light (Mr 15,000) subunits. To study the assembly of dimeric myeloperoxidase azurophilic granules were isolated from either unlabeled or pulse-labeled ([35S]methionine/cysteine) HL-60 cells, and myeloperoxidase was extracted and separated into monomeric and dimeric forms by FPLC gel filtration chromatography. Steady-state levels of dimeric and monomeric myeloperoxidase were found to account for 67% and 33%, respectively, of the total peroxidase activity and were correlated with the levels of associated heme as measured by absorption at 430 nm. Labeled myeloperoxidase polypeptides were immunoprecipitated using a monospecific rabbit antibody and were identified and quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography and liquid scintillation counting. After a 2-h pulse, labeled myeloperoxidase species of Mr 74,000 and 60,000 were found in fractions coeluting with the monomeric form of myeloperoxidase. Following chase periods from 6 to 68 h, 18-70%, respectively, of the total immunoprecipitable, labeled myeloperoxidase moved out of the monomer pool and was found to be associated with a Mr 60,000 subunit that coeluted with the dimeric form of myeloperoxidase. Quantitation of the time course of the conversion of monomeric to dimeric myeloperoxidase indicated a precursor-product relationship at the level of the mature Mr 60,000 subunit. The assembly of dimeric enzyme is a relatively late event in maturation with a t1/2 of 36 h, implying that this process occurs in more mature, dense azurophilic granules.