The post-translational processing of myeloperoxidase is regulated by the availability of heme.

Myeloperoxidase (MPO) is a hemoprotein that is synthesized in the lumen of the endoplasmic reticulum (ER) as a single-chain precursor and undergoes a complex series of post-translational modifications prior to packaging into azurophilic granules. We and others have previously observed that treatment of human myeloid leukemic cells with succinylacetone (SA), a potent inhibitor of 5-aminolevulinic acid dehydratase (ALA-D), and hence of heme biosynthesis, resulted in loss of MPO enzyme activity, inhibition of the appearance of mature MPO, and accumulation of enzymatically unreactive, but immunoreactive, MPO in the ER. The present study using HL-60 cells was undertaken to establish the nature and specificity of the inhibition by SA and to identify and quantify the biochemical changes in the post-translational pathway of MPO processing. Dose-response studies showed that SA (250 microM) did not affect cell viability or growth up to 72 h, but resulted in inhibition of ALA-D activity (> 93%) and decreased cellular levels of both heme and MPO (approximately 25% of control). There were no effects on the level of total cellular protein or on the activities of lactate dehydrogenase or several other nonheme enzymes colocalized with MPO in azurophilic granules. Northern blot analyses confirmed the nontoxic nature of the conditions and indicated there was no effect on transcription of MPO mRNA. The kinetics of processing in the presence and absence of 250 microM SA were determined using pulse-chase and Percoll density gradient centrifugation methods, followed by identification and quantification of MPO species by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The initial rate of disappearance of precursor MPO was identical for control and SA-treated cells and, after a lag of 2-3 h, there was a fourfold decrease in the rate of appearance of mature MPO in SA-treated cells. In the presence of SA, precursor apoMPO remained in the ER, did not undergo proteolytic processing and, compared to control cells, about 50% was degraded. The disruption in MPO processing was reversible by the addition of exogenous heme. We conclude that the availability of heme is important in the complex maturation of MPO that occurs in the ER, events which precede exit from this compartment and subsequent proteolytic processing and transport to the azurophilic granule.

Ultrastructural, immunochemical, and cytochemical study of myeloperoxidase in myeloid leukemia HL-60 cells following treatment with succinylacetone, an inhibitor of heme biosynthesis.

Myeloperoxidase (MPO) is a heme-containing glycoprotein found in the primary granules (or azurophilic granules) of human polymorphonuclear leukocytes. In the present study, cultured myeloid leukemia HL-60 cells were exposed for 0-72 h to 250 microM 4,6-dioxoheptanoic acid (succinylacetone, SA), a specific inhibitor of heme biosynthesis, and the effects were evaluated using ultrastructural, immunochemical, and cytochemical methods. En bloc peroxidase staining of glutaraldehyde-fixed cells was accomplished with a 30-min exposure to 3,3'-diaminobenzidine (DAB) tetrahydrochloride. Ultrastructural examination revealed that peroxidase reactivity in the endoplasmic reticulum (ER) was relatively unchanged for 8 h and decreased between 12 and 24 h; however, ER lacked DAB-reactive peroxidase at 48-72 h. Peroxidase reactivity in the ER reappeared within 4 h after removal of SA. Seventy-two hours after exposure to SA the number of condensed cytoplasmic granules stained with DAB was significantly decreased, and many of the granules had a "target" appearance with a central DAB-reactive dense core. Staining of mitochondria was observed with overnight exposure to DAB and persisted in HL-60 cells treated 72 h with SA. Mitochondrial and nuclear morphology appeared unaltered. Immunostaining of MPO in thin sections of paraformaldehyde/glutaraldehyde-fixed unosmicated HL-60 cells, embedded in Lowicryl K4M, was accomplished with sequential exposure to an affinity-purified monospecific rabbit antibody to HL-60-MPO and protein A conjugated to 5- or 10-nm colloidal gold. Compared to untreated control HL-60 cells, cells exposed to SA for 48 h exhibited comparable to increased immunoreactive MPO in the ER, despite the absence of heme-dependent peroxidase reactivity. The data indicate that SA inhibits formation of enzymatically active MPO and that in the presence of SA, the ER contains a form(s) of MPO that lacks enzymatic reactivity.

Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity.

The enzyme myeloperoxidase (MPO) is a functionally important glycoprotein of neutrophilic granulocytes and occurs in three major isoforms (forms 1, 2, and 3) that are dimeric structures composed of two heavy subunit-light subunit protomers, each of which is associated with a chlorine-like prosthetic group. In the present study, highly purified MPO isoforms were obtained from the cells of a single normal donor, and each protein was subjected to reductive alkylation under nondenaturing conditions. The resulting enzymatically active protomers were separated from unreacted dimer using gel filtration chromatography. Use of a fast protein liquid chromatography cation exchange system with a Mono S matrix revealed heterogeneity of the protomers, and allowed essentially complete resolution of the protomers of MPO form 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two resolved protomeric species under reducing conditions revealed small but reproducible differences in the Mr of their heavy subunits (59,000 and 57,000). Treatment with either endo-beta-N-acetylglucosaminidase or peptide N-glycohydrolase F reduced the Mr of each heavy subunit by approximately 3000 but did not change their relative electrophoretic mobilities. Heavy and light subunits were prepared from each of the MPO isoforms by reductive alkylation under conditions that allowed full retention of the prosthetic group with the heavy subunit. Reverse-phase chromatography and amino-terminal sequencing showed that each MPO isoform contained one major species of light subunit and several minor species. No differences in peroxidatic activity or inhibition by salicylhydroxamic acid were observed among any of the MPO isoforms or resolved protomers, but the latter were considerably more heat labile than dimeric forms of the enzyme and a monomeric form isolated from HL-60 cells. This is the first report of the isolation and partial characterization of distinct protomers from a single isoform of human MPO and suggests that the structure of MPO is more complex than considered previously.

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

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

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)

Monensin disruption of neutrophil granule genesis.

The Na+/H+ ionophore monensin (M) has been used widely to study intracellular pH gradients and acidic subcellular compartments. In the present study, cultured myeloid leukemia HL60 cells, directly sampled bone marrow cells, and peripheral blood neutrophils were exposed to 1-5 microM monensin for 0.5-20 hours. The effects were evaluated using ultrastructural, cytochemical, and biochemical methods. In HL60 cells and marrow promyelocytes treated with monensin, progressive vacuolation of the trans then the cis Golgi was observed. These vacuoles lacked diaminobenzidine (DAB) reactive peroxidase, high iron diamine (HID) reactive sulfated glycoconjugates, and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reactive vicinal glycol containing complex carbohydrates, but some cis Golgi elements retained osmium zinc iodide reactive reducing groups. The number of normal intensely stained HID reactive granules decreased and an incomplete granule that was DAB-positive/HID-negative, PA-TCH-SP-negative with flocculent matrix density increased in frequency as a function of time and concentration of monensin. Treatment of HL60 cells with monensin markedly reduced 35SO4 incorporation but myeloperoxidase labeling and activity per cell remained constant, although it shifted to lower density granule fractions consistent with the persistent DAB staining of endoplasmic reticulum and synthesis of a DAB-positive, HID-negative granule in intact HL60 cells. The Golgi complex of monensin-treated myelocytes and segmented neutrophils was also vacuolated. A subpopulation of preformed primary granules in promyelocytes, myelocytes, and segmented neutrophils appeared to increase in size and peripheral or central electron lucency. These selective effects of monensin indicate that granule components may be packaged into DAB-positive organelles that are deficient in trans Golgi-derived elements (HID- and PA-TCH-SP-negative) and that some preformed primary granules contain a monensin sensitive Na+/H+ gradient.

A monoclonal antibody that inhibits the antimicrobial action of a 57 KD cationic protein of human polymorphonuclear leukocytes.

Two monoclonal antibodies (mAb) specific for epitopes of a 57,000 m.w., cationic antimicrobial protein (CAP57) purified from granules of human polymorphonuclear leukocytes (PMN) have been produced. Both were IgG1 mouse antibodies with typical heavy and light chain structure. The mAb reactive with CAP57 failed to react specifically with other heretofore defined PMN or serum proteins as shown by ELISA. Both mAb showed specific reactivity in Western blots with CAP57. One of these mAb (P1G8) inhibited the antimicrobial action of CAP57 by 50% at a ratio of 62.5 micrograms antibody per microgram CAP57. The other mAb, P2A5, had no inhibitory capacity for CAP57. Binding constants of the two mAb for the antigen were determined and were found to be virtually identical. Thus, the greater inhibitory capacity of P1G8 for bacterial killing by CAP57 was not directly related to binding strength of the mAb. Competition experiments showed that unlabeled P1G8 could compete as well against radiolabeled P2A5 as could unlabeled P2A5. In the reverse experiment, it was seen that P1G8 competed with radiolabeled P1G8 for CAP57 better than unlabeled P2A5. These findings could be due to two antibodies that recognize different but adjacent epitopes on CAP57, one of the epitopes (P1G8) being closer to structure(s) of the protein essential to its antimicrobial action. Immunocytochemical studies showed positive staining with both mAb. The reaction was restricted to the cytoplasm of peripheral blood PMN and was of a granular pattern. Other peripheral blood cells (which included red blood cells, eosinophils, monocytes, and lymphocytes) failed to bind either mAb.

Peritoneal macrophages of pathogen-free rats but not of conventional rats secrete elastolytic activity.

Elicited peritoneal macrophages from Sprague-Dawley rats conventionally bred and housed failed, as we have reported, to produce detectable elastolytic activity in culture. They did produce lysozyme and plasminogen activator. We now show that in contrast to these cells, macrophages from pathogen-free, barrier-sustained rats produced readily demonstrable elastolytic activity. Rats raised pathogen-free and subsequently housed conventionally for 2-4 wk appeared to lose the capacity to afford macrophages producing elastase. At the same time they acquired infections with several rat pathogens including Spironucleus muris, Kilham rat virus, sialodacryoadinitis virus, and mycoplasma pulmonis. The acquisition by the rats of one or more of these infections, conditions conducive to infection, or both factors may have suppressed their capacity to yield elastolytic activity.