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1.
J Biochem ; 161(1): 79-86, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27650603

RESUMO

The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4 Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4 The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.


Assuntos
Afinidade de Anticorpos/genética , Leucotrieno E4/química , Mutação de Sentido Incorreto , Anticorpos de Cadeia Única/química , Substituição de Aminoácidos , Animais , Camundongos , Anticorpos de Cadeia Única/genética
3.
Biochim Biophys Acta ; 1840(6): 1625-33, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24361619

RESUMO

BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.


Assuntos
Anticorpos Monoclonais/farmacologia , Leucotrieno C4/imunologia , Leucotrieno D4/imunologia , Anticorpos de Cadeia Única/farmacologia , Animais , Anticorpos Monoclonais/uso terapêutico , Asma/tratamento farmacológico , Células CHO , Cricetinae , Cricetulus , Citocinas/biossíntese , Humanos , Antagonistas de Leucotrienos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Receptores de Leucotrienos/efeitos dos fármacos , Receptores de Leucotrienos/fisiologia
4.
Biochem Biophys Res Commun ; 392(3): 421-5, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20079714

RESUMO

Leukotriene C(4) (LTC(4)) is synthesized by binding of glutathione to LTA(4), an epoxide derived from arachidonic acid, and further metabolized to LTD(4) and LTE(4). We previously prepared a monoclonal antibody with a high affinity and specificity to LTC(4). To explore the structure of the antigen-binding site of a monoclonal antibody against LTC(4) (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC(4) comparable to mAbLTC. The scFvLTC also bound to LTD(4) and LTE(4) with 48% and 17% reactivities, respectively, as compared with LTC(4) binding, whereas the antibody showed almost no affinity for LTB(4).


Assuntos
Anticorpos Monoclonais/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Leucotrieno C4/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Clonagem Molecular , DNA Complementar/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
5.
Luminescence ; 24(2): 131-3, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19291811

RESUMO

Prostaglandin E(2) is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E(2) utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E(2) amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E(2) required to displace 50% of the maximal binding of Cypridina luciferase-labeled prostaglandin E(2) (B/B(0)) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme-linked immunosorbent assay.


Assuntos
Dinoprostona/análise , Técnicas Imunoenzimáticas/métodos , Luciferases , Animais , Cyprinidae , Humanos , Substâncias Luminescentes , Medições Luminescentes
6.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 64(Pt 11): 1027-30, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18997333

RESUMO

Prostaglandin E(2) is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E(2) is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E(2) antibody was prepared and its complex with prostaglandin E(2) was crystallized. The stable Fab-prostaglandin E(2) complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 A. The asymmetric unit was suggested to contain one molecule of the Fab-prostaglandin E(2) complex, with a corresponding crystal volume per protein weight of 2.75 A(3) Da(-1).


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Dinoprostona , Fragmentos Fab das Imunoglobulinas/química , Animais , Anticorpos Monoclonais/imunologia , Cristalização , Cristalografia por Raios X , Dinoprostona/química , Dinoprostona/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Ligantes , Camundongos , Estrutura Molecular , Difração de Raios X
7.
Biochem Biophys Res Commun ; 367(4): 782-6, 2008 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-18198127

RESUMO

Membrane-associated prostaglandin (PG) E synthase (mPGE synthase)-2 catalyzes the conversion of PGH(2) primarily to PGE(2). The enzyme is activated by various sulfhydryl reagents including dithiothreitol, dihydrolipoic acid, and glutathione, and it is different from mPGE synthase-1 and cytosolic PGE synthase, both of which require specifically glutathione. Recently, other investigators reported that their preparation of mPGE synthase-2 containing heme converted PGH(2) to 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) rather than to PGE(2) [T. Yamada, F. Takusagawa, Biochemistry 46 (2007) 8414-8424]. As we examined presently, the heme-bound enzyme expressed and purified according to their method synthesized HHT from PGH(2), but also PGE(2) in a decreased amount. Whereas the PGE synthase activity was completely lost at 50 degrees C for 5 min, the HHT synthase activity remained even at 100 degrees C for 5 min. In contrast, when the heme-bound enzyme was purified in the presence of dithiothreitol, only PGE(2) was produced, but essentially no HHT was detected. Thus, native mPGE synthase-2 enzymatically catalyzes only the conversion of PGH(2) to PGE(2), but not to HHT, and heme is not involved in this reaction.


Assuntos
Membrana Celular/metabolismo , Ditiotreitol/metabolismo , Escherichia coli/metabolismo , Harringtoninas/metabolismo , Oxirredutases Intramoleculares/metabolismo , Escherichia coli/genética , Prostaglandina-E Sintases , Proteínas Recombinantes/metabolismo
9.
Mol Carcinog ; 45(4): 250-9, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16385588

RESUMO

Considering possible tumorigenic activity of cyclooxygenase (COX) isozymes in myeloma, we examined expression levels of COX-1 and -2 in seven human myeloma cell lines (ARH-77, IM-9, RPMI-8226, HPC, HS-Sultan, TSPC-1, and U-266). As analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), all the cell lines constitutively expressed COX-1, while COX-2 levels markedly varied among different cell lines. Induction of COX-2 by phorbol ester was observed in RPMI-8226 and HPC cells. In contrast, COX-2 was constitutively expressed in ARH-77 and IM-9 cells. Moreover, the high expression level of COX-2 protein in ARH-77 cells was verified by Western blotting. Intact cells of ARH-77 converted 14C-labeled arachidonic acid to prostaglandin E2, F2alpha, and D2, and this activity was dose-dependently inhibited by selective COX-2 inhibitors (SC-58125 and NS-398), a non-selective COX inhibitor (indomethacin), and relatively high concentrations of a selective COX-1 inhibitor (SC-560). These COX inhibitors also suppressed the proliferation of ARH-77 cells, but significant suppression was seen only at 100 microM, a much higher concentration than those sufficient for the COX inhibition. Moreover, proliferation of the myeloma cells lacking COX-2 was also suppressed by 100 microM of SC-58125. These results suggested that the anti-proliferative effect of the COX inhibitors is independent of the inhibition of COX-2.


Assuntos
Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Mieloma Múltiplo/enzimologia , Ácido Araquidônico/metabolismo , Western Blotting , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Ésteres de Forbol/farmacologia , Prostaglandina D2/metabolismo , Pirazóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia
10.
Biochem Biophys Res Commun ; 338(1): 122-7, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16171776

RESUMO

Lipoxygenase is a dioxygenase recognizing a 1-cis,4-cis-pentadiene of polyunsaturated fatty acids. The enzyme oxygenates various carbon atoms of arachidonic acid as a substrate and produces 5-, 8-, 12- or 15-hydroperoxyeicosatetraenoic acid with a conjugated diene chromophore. The enzyme is referred to as 5-, 8-, 12- or 15-lipoxygenase, respectively. Earlier we found two isoforms of 12-lipoxygenase, leukocyte- and platelet-type enzymes, which were distinguished by substrate specificity, catalytic activity, primary structure, gene intron size, and antigenicity. Recently, the epidermis-type enzyme was found as the third isoform. Attempts have been made to find isozyme-specific inhibitors of 12-lipoxygenase, and earlier we found hinokitiol, a tropolone, as a potent inhibitor selective for the platelet-type 12-lipoxygenase. More recently, we tested various catechins of tea leaves and found that (-)-gallocatechin gallate was a potent and selective inhibitor of human platelet 12-lipoxygenase with an IC50 of 0.14 microM. The compound was much less active with 12-lipoxygenase of leukocyte-type, 15-, 8-, and 5-lipoxygenases, and cyclooxygenases-1 and -2.


Assuntos
Ácido Araquidônico/metabolismo , Inibidores de Lipoxigenase , Animais , Araquidonato 12-Lipoxigenase/química , Araquidonato 12-Lipoxigenase/genética , Ácido Araquidônico/química , Catequina/análogos & derivados , Catequina/farmacologia , Humanos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Suínos
11.
Free Radic Biol Med ; 34(3): 304-15, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12543246

RESUMO

Lipoxygenases (LOXs) are multifunctional enzymes that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives; they also convert hydroperoxy fatty acids to epoxy leukotrienes and other secondary products. LOXs undergo suicidal inactivation but the mechanism of this process is still unclear. We investigated the mechanism of suicidal inactivation of the rabbit 15-lipoxygenase by [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid (15-HpETE) and observed covalent modification of the enzyme protein. In contrast, nonlipoxygenase proteins (bovine serum albumin and human gamma-globulin) were not significantly modified. Under the conditions of complete enzyme inactivation we found that 1.3 +/- 0.2 moles (n = 10) of inactivator were bound per mole lipoxygenase, and this value did depend neither on the enzyme/inactivator ratio nor on the duration of the inactivation period. Covalent modification required active enzyme protein and proceeded to a similar extent under aerobic and anaerobic conditions. In contrast, [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE), which is no substrate for epoxy-leukotriene formation, did not inactivate the enzyme and protein labeling was minimal. Separation of proteolytic cleavage peptides (Lys-C endoproteinase digestion) by tricine SDS-PAGE and isoelectric focusing in connection with N-terminal amino acid sequencing revealed covalent modification of several active site peptides. These data suggest that 15-lipoxygenase-catalyzed conversion of (15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid to 14,15-epoxy-leukotriene leads to the formation of reactive intermediate(s), which are covalently linked to the active site. Therefore, this protein modification contributes to suicidal inactivation.


Assuntos
Araquidonato 15-Lipoxigenase/química , Araquidonato 15-Lipoxigenase/metabolismo , Leucotrienos/farmacologia , Peróxidos Lipídicos/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Leucotrienos/metabolismo , Peróxidos Lipídicos/metabolismo , Modelos Moleculares , Desnaturação Proteica , Estrutura Secundária de Proteína , Coelhos
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