Spectral Analysis of Lactoperoxidase

The seven members of the NOX/DUOX family are responsible for generation of the superoxide and H 2 O 2 required for a variety of host defense and cell signaling functions in nonphagocytic cells. Two members, the dual oxidase isozymes DUOX1 and DUOX2, share a structurally unique feature: an N-terminal peroxidase-like domain. Despite sequence similarity to the mammalian peroxidases, the absence of key active site residues makes their binding of heme and their catalytic function uncertain. To explore this domain we have expressed in a baculovirus system and purified the Caenorhabditis elegans (CeDUOX1 1-589 ) and human (hDUOX1 1-593 ) DUOX1 "peroxidase" domains. Evaluation of these proteins demonstrated that the isolated hDUOX1 1-593 does not bind heme and has no intrinsic peroxidase activity. In contrast, CeDUOX1 1-589 binds heme covalently, exhibits a modest peroxidase activity, but does not oxidize bromide ion. Surprisingly, the heme appears to have two covalent links to the protein despite the absence of a second conserved carboxyl group in the active site. Although the N-terminal dual oxidase motif has been proposed to directly convert superoxide to H 2 O 2 , neither DUOX1 domain demonstrated significant superoxide dismutase activity. These results strengthen the in vivo conclusion that the CeDUOX1 protein supports controlled peroxidative polymerization of tyrosine residues and indicate that the hDUOX1 protein either has a unique function or must interact with other protein factors to express its catalytic activity.

Section: Design and Expression Of Soluble Duox1mentioning

confidence: 91%

Caenorhabditis elegans and Human Dual Oxidase 1 (DUOX1) “Peroxidase” Domains

Meitzler

Montellano

2009

“…As mentioned above, TPO, LPO, MPO, and EPO belong to the mammalian peroxidase family. Spectral analysis (12) indicated that the heme of TPO could be a type l heme like LPO. Moreover, Kooter et al (27) also indicated that the prosthetic group of mammalian peroxidase was 1,5-dihydroxymethyl-modified heme b attached by covalent bonds to the protein.…”

Section: Resultsmentioning

confidence: 99%

“…In mammalian peroxidases, the heme moiety is covalently linked to the protein backbone (11,12). As mentioned above, TPO, LPO, MPO, and EPO belong to the mammalian peroxidase family.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, their prosthetic heme moieties are not readily extracted by conventional approaches. Anderson et al (12) considered that spectral similarities between LPO and other mammalian peroxidases indicated similar prosthetic heme moieties and a covalent attachment of the heme group to the protein. They suggested that the heme moiety of TPO is probably a type l heme, like LPO.…”

Section: Thyroperoxidase (Tpo)mentioning

confidence: 99%

See 1 more Smart Citation

Role of Heme in Intracellular Trafficking of Thyroperoxidase and Involvement of H2O2 Generated at the Apical Surface of Thyroid Cells in Autocatalytic Covalent Heme Binding

Fayadat¹,

Niccoli-Sire

Lanet

et al. 1999

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by ϳ30 and ϳ80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (؉120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H 2 O 2 -dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H 2 O 2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 M H 2 O 2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H 2 O 2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H 2 O 2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule. Thyroperoxidase (TPO)1 is a membrane-bound, glycosylated hemoprotein that plays a key role in the biosynthesis of thyroid hormones. It catalyzes the iodination of thyroglobulin and the coupling of some of the iodotyrosyl residues to produce thyroxine and 3,3Ј,5-triiodothyronine (1-3 (2) showed significant differences between the pyridine hemochromogene of TPO and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. TPO, lactoperoxidase (LPO), myeloperoxidase (MPO), saliva peroxidase, eosinoperoxidase, and intestinal peroxidase belong to the mammalian peroxidase family. These proteins share related protein primary structure (10, 11) and are alike in spectral properties. Furthermore, their prosthetic heme moieties are not readily extracted by conventional approaches. Anderson et al. (12) considered that spectral similarities between LPO and other mammalian peroxidases indicated similar prosthetic heme moieties and a covalent attachment of the heme group to the protein. They suggested that the heme moiety of TPO is probably a type l heme, like LPO. This heme is covalently linked to the protein through ester bonds that link aspartate and glutamate residues (conserved in MPO, ...

“…posed previously (20)), but from noncovalent interactions within the protein milieu. Of the noncovalently bound heme, the majority is unmodified iron protoporphyrin IX (Fig.…”

Section: Autocatalytic Processing Of Heme 8858mentioning

confidence: 99%

Autocatalytic Processing of Heme by Lactoperoxidase Produces the Native Protein-bound Prosthetic Group

DePillis

Ozaki

Kuo

et al. 1997

104

The covalently bound prosthetic group of lactoperoxidase (LPO) has been obtained by hydrolysis of the protein and identified as a dihydroxylated heme. A baculovirus expression system has been developed for LPO and used to obtain protein in which the heme is only partially covalently bound. Reaction of the purified heme⅐apoLPO complex with H 2 O 2 results in both autocatalytic modification of the heme and covalent attachment to the protein. Hydrolytic experiments establish that the autocatalytically incorporated heme is bound normally. Two monohydroxylated heme intermediates have been detected. The peroxidative activity of LPO increases in proportion to the extent of covalently bound heme. The LPO results provide a paradigm for autocatalytic incorporation of heme groups into the mammalian peroxidases, including myeloperoxidase and eosinophil peroxidase, all of which exhibit strong sequence similarity with LPO and have covalentlybound heme groups.The mammalian peroxidases, including lactoperoxidase (LPO), 1 myeloperoxidase (MPO), eosinophil peroxidase, and thyroid peroxidase, utilize H 2 O 2 to catalyze a diverse set of reactions. LPO (1), MPO (2), and eosinophil peroxidase (3) contribute to the nonimmune host defense system by oxidizing chloride ion and the pseudohalide thiocyanate to the potent microbicidal agents hypochlorous and hypothiocyanous acids, respectively. LPO fulfills this function in exocrine secretions, including tears, milk, and saliva, while eosinophil peroxidase and MPO carry out this chemistry in the phagosomes of neutrophils and eosinophils during engulfment of microorganisms. Thyroid peroxidase, which is distinguished from the other peroxidases in that it is an intracellular membrane-bound protein, catalyzes the iodination and coupling of thyroglobulin moieties in the biosynthesis of the thyroid hormones thyroxine and triiodothyronine (4). The mammalian peroxidases also participate in the oxidative metabolism of xenobiotics responsible for hypersensitivity reactions and other toxic sequelae (5, 6).The ease with which the mammalian peroxidases oxidize high potential substrates such as pseudohalides distinguishes them from enzymes such as horseradish peroxidase and yeast cytochrome c peroxidase. The oxidative potency of the mammalian enzymes may be related to one of their distinguishing features: the presence of a modified, covalently bound heme group. The prosthetic group of MPO has been identified by crystallographic and chemical studies as a 1,5-dihydroxymethyl-modified heme b attached by three bonds to the protein (7-9). Two of these are ester bonds that link Asp and Glu residues with the heme hydroxymethyl groups, and the third is a sulfonium ion link obtained by addition of a Met sulfur atom to the 2-vinyl moiety of the heme. The structure of the heme group in the other mammalian peroxidases and the mechanism by which the hemes are incorporated into the mature proteins remain unknown. EXPERIMENTAL PROCEDURESCloning and Expression-The cDNA for bovine LPO (10) was subcloned into pAcGP67B us...