The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144,111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 pg * ml-' * day-'. The recombinant product was purified by a combination of ion-exchange and metalchelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Aminoterminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichiu coli when provided with its physiological substrates, i. e. hydrogen peroxide and chloride ions.
Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killed Candida albicans at a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and that C. albicans is less resistant to MPO-dependent oxidants than less pathogenic Candida species. We hypothesized, therefore, that the capacity of macrophages to kill C. albicans might be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing of C. albicans by resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonized C. albicans resulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whether C. albicans was opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing of C. albicans by monocytes, nor did it affect phagocytosis of opsonized or unopsonized C. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis.
A chimeric antibody-like molecule consisting of the human myeloperoxidase (rMPO) fused to the second and third constant-sequence (CH2 and CH3) Fc domains of human immunoglobulin G-1 has been constructed and expressed in Chinese hamster ovary (CHO) cells. This fusion molecule was designed to combine the binding specificity of Fc with the antimicrobial properties of rMPO. The rMPO-Fc fusion dimerized through the Fc fragment, while retaining the enzymatic activity of rMPO. The chimeric molecule was glycosylated and most of the propeptide was eliminated, indicating a better processing of the polypeptide than for rMPO alone. Both rMPO and rMPO-Fc bound to and were internalized by macrophage-like U937 promonocytic cells. Unexpectedly, the chimera failed to bind to the Fc receptor but interacted with a higher affinity than rMPO with the same binding sites. The presence of the Fc fragment in the chimera, in addition, did not extend the plasma half-life of the fusion protein. In vitro, rMPO-Fc exhibited a stronger killing effect than rMPO toward Candida albicans in the presence of either H202 alone or human macrophages. In vivo, rMPO-Fc similarly conferred a better protection than rMPO in a lethal model of murine cowdriosis. These properties could be related to the Fc-induced dimerization of the fusion protein in CHO cells.
The effect of the human recombinant myeloperoxidase (recMPO) system on the viability of Candida albicans blastospores was determined. Cells were incubated in the presence of the enzyme, H202, and various substrates. In optimal conditions, 100% mortality was reached with only 41 mU of recMPO per ml, confirming its powerful candidicidal activity.To fight invading microorganisms, polymorphonuclear neutrophils use essentially two mechanisms. The first one, which is nonoxidative, involves such molecules as defensins, permeability-inducing proteins, proteases, or lysozyme (12). The second process occurs via the respiratory burst; this oxygen-dependent system leads to the production of reactive oxygen metabolites, such as H202 (reviewed in reference 5). The toxicity of the generated hydrogen peroxide is increased by reaction with a granular enzyme called myeloperoxidase (MPO) and a halide to form potent halide oxidation products such as hypochlorous acid (HOCl) (9).The candidicidal activity of the natural MPO system has been documented (6, 11); it has been shown also that MPO-deficient polymorphonuclear neutrophils lose their candidicidal property (4) and that in monocytes maturing into macrophages, the decrease in MPO content is accompanied by a decline in candidicidal activity (13). Moreover, a few MPO-deficient individuals have developed serious hematogenous Candida infections (15).In view of the potential interest of MPO as an antifungal agent, we studied the effects of a recombinant form of the enzyme (recMPO) (14) on the viability of Candida albicans blastospores, using as substrates H202 generated by glucose and glucose oxidase and physiological (Cl-) and (I-) halides. In addition, the candidicidal activity of the recMPO system was evaluated at different pHs and in the presence of ammonium ions, which by reaction with HOCl yields strongly oxidizing monochloramines (15).Candidicidal system. Experiments were conducted with a C. albicans strain, 10231, obtained from the American Type Culture Collection. Cells were grown on YM (Difco) agar slopes for 30 h at 30°C and then collected by rinsing with 1 ml of 0.9% NaCl. The suspension was centrifuged and resuspended in 20 mM NaKPO4 buffer, pH 7.5, to a density of 106 cells per ml. For each assay, 1 ml of the suspension was incubated with gentle stirring for 60 min at 37°C, with the various components of the reaction system (i.e., recMPO, glucose, and glucose oxidase) and a halide, in a final volume of 5 ml of NaKPO4 buffer, pH 7.5. Aliquots of the reaction mix were collected, diluted with 0.9% NaCl and spread in triplicate onto Sabouraud dextrose agar plates containing 50 ,ug of chloramphenicol per ml. Colonies were counted after incubation of the plates for 48 h at 30°C.An initial control experiment determined the maximal * Corresponding author.H202 concentration tolerated by C. albicans blastospores; to this end, the concentration of glucose was maintained at 1%while the concentration of glucose oxidase was varied. As shown in Fig. 1, catalase-positive C. albicans sup...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.