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Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells
K L Taylor1, G S Guzman, C A Burgess
1Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Myeloperoxidase (MPO) in neutrophils exists as dimers in mature cells and monomers/dimers in leukemia cells. This study investigates MPO assembly, revealing precursor processing and subunit association in host defense.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Myeloperoxidase (MPO) is crucial for neutrophil bactericidal activity.
- MPO exists as a dimer in mature neutrophils but shows monomeric and dimeric forms in leukemia cells.
- MPO is synthesized as a precursor that is processed into mature subunits.
Purpose of the Study:
- To investigate the assembly process of dimeric myeloperoxidase.
- To analyze the forms of MPO present in HL-60 leukemia cells.
- To understand the relationship between MPO forms, heme association, and precursor processing.
Main Methods:
- Isolation of azurophilic granules from HL-60 cells (pulse-labeled with [35S]methionine/cysteine).
- Extraction and separation of monomeric and dimeric MPO using FPLC gel filtration chromatography.
- Immunoprecipitation of labeled MPO polypeptides followed by SDS-PAGE/fluorography and liquid scintillation counting.
Main Results:
- Steady-state MPO activity comprises 67% dimeric and 33% monomeric forms, correlated with heme levels.
- Pulse-labeling revealed Mr 74,000 and 60,000 MPO species co-eluting with the monomeric form after 2 hours.
- This suggests the involvement of these processed forms in MPO monomer assembly.
Conclusions:
- The study elucidates the processing and assembly of myeloperoxidase in neutrophils.
- Immature MPO forms are identified and associated with the monomeric species.
- Findings contribute to understanding MPO's role in host defense and its alterations in leukemia.