Studies on the subunits of human myeloperoxidase

Olsen, R L; Little, C. H. A.

doi:10.1042/bj2220701

Cited by 72 publications

(35 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…3B shows the analysis of intact and H 2 O 2 -treated MPO, EPO, and LPO on a 20% SDS-polyacrylamide gel. As known (38,39), both native MPO and native EPO appeared on SDS-PAGE as two bands, one of approximately 50 to 60 kDa and the other of 10.5 to 15 kDa. However, neither MPO nor EPO gave a new protein band when treated with H 2 O 2 .…”

Section: Spin Trapping Of Lpo Protein-derived Radicals By Db-nbs-mentioning

confidence: 99%

Spin Trapping and Protein Cross-linking of the Lactoperoxidase Protein Radical

Lardinois

Medzihradszky

Montellano

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Lactoperoxidase (LPO) reacts with H 2 O 2 to sequentially give two Compound I intermediates: the first with a ferryl (Fe IV ‫؍‬O) species and a porphyrin radical cation, and the second with the same ferryl species and a presumed protein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid to give a 1:1 protein-bound radical adduct. Furthermore, LPO undergoes the H 2 O 2 -dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in each of two LPO molecules. The dimer retains full catalytic activity and reacts to the same extent with the spin trap, indicating that the spin trap reacts with a radical center other than Tyr-289. The monomeric protein recovered from incubations of LPO with H 2 O 2 is fully active but no longer forms dimers when incubated with H 2 O 2 , clear evidence that it has also been structurally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H 2 O 2 -dependent oligomerization. Analysis of the interface in the LPO dimers indicates that the same protein surface is involved in LPO dimerization as in the normal formation of myeloperoxidase dimers. Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates. LPO1 (EC 1.11.1.7; donor-H 2 O 2 oxidoreductase), which oxidizes thiocyanate (HSCN) to hypothiocyanate (HOSCN) and more highly oxidized species, is a component of the mammalian antimicrobial defense system (1). It is closely related by sequence and function to MPO and EPO and also by sequence to thyroid peroxidase (2, 3). LPO is not only of intrinsic interest but is also a useful model for the study of MPO and EPO, neither of which has been successfully expressed in other than mammalian cells (4).Bovine LPO is a protein of 612 amino acids with a molecular mass of approximately 78,000 Da, approximately 10% of which is carbohydrate (2, 5). The prosthetic group of the enzyme is a heme in which the 1-and 5-methyl groups bear hydroxyl groups esterified with the side chains of respectively (4,[6][7][8]. The prosthetic heme in myeloperoxidase is bound via two similar ester bonds to the protein (9) but in addition is linked to the protein by a bond between the 2-vinyl group and a methionine residue (10, 11). We established earlier that the prosthetic group is bound to the protein in LPO via a self-activating process in which noncovalently bound heme becomes covalently bound on exposure of the heme-protein complex to H 2 O 2 (4). Recent evidence indicates that a similar mechanism is involved in covalent attachment of the heme to the protein in EPO and thyroid peroxidase (12, 13).The peroxidative mechanism for LPO, as is true for all hemoprotein peroxidases, involves the following three-step sequence, where AH is a substrate (14).

show abstract

Section: Spin Trapping Of Lpo Protein-derived Radicals By Db-nbs-mentioning

confidence: 99%

Spin Trapping and Protein Cross-linking of the Lactoperoxidase Protein Radical

Lardinois

Medzihradszky

Montellano

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The synthesis of MPO, in vivo, occurs at the promyelocytic stage of the myeloid differentiation pathway (Bainton & Farquhar, 1966). It starts with the production of a glycosylated 89-kDa precursor protein (Yamada et al, 1982(Yamada et al, , 1990 which is subsequently processed via several intermediates into heavy and light chains of 60 kDa and 15 kDa (Olsen & Little, 1984;Akin & Kinkade, 1986;Nauseef & Clark, 1986). The mature enzyme, which carries two identical prosthetic groups, is a 150-kDa dimer made of one heavy and one light chain (Andrew & Krinsky, 1981).…”

mentioning

confidence: 99%

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines

Moguilevsky

Garcia‐Quintana

Jacquet

et al. 1991

European Journal of Biochemistry

124

View full text Add to dashboard Cite

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.

show abstract

“…In contrast, the polypeptide chains of MPO and EPO are cleaved after synthesis to form a heavy chain of approximately 55 kDa and a light chain of approximately 15 kDa that remain associated. EPO is a monomer with the expected size of 70 kDa, whereas MPO is a disulfide-linked dimer of 150 kDa (12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

Biochemical Evidence for Heme Linkage through Esters with Asp-93 and Glu-241 in Human Eosinophil Peroxidase

Oxvig

Thomsen

Overgaard

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.

show abstract

Studies on the subunits of human myeloperoxidase

Cited by 72 publications

References 33 publications

Spin Trapping and Protein Cross-linking of the Lactoperoxidase Protein Radical

Spin Trapping and Protein Cross-linking of the Lactoperoxidase Protein Radical

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines

Biochemical Evidence for Heme Linkage through Esters with Asp-93 and Glu-241 in Human Eosinophil Peroxidase

Contact Info

Product

Resources

About