Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Human M-ficolin is a secretory protein that activates the lectin complement pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a co-immunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.     

Simultaneous resonance Raman detection of the heme a3-Fe-CO and CuB-CO species in CO-bound ba3-cytochrome c oxidase from Thermus thermophilus. Evidence for a charge transfer CuB-CO transition

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.     

Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase

Reduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron-oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with Cu(B) and the rate of the reaction from Oxy to P (P(R)) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fe(a(3))-O-O-Cu(B) peroxy bridged structure. The Fe(a(3))=O stretching (nu(Fe=O)) frequency of the P(R) intermediate, 804/764 cm(-1) for (16)O/(18)O, is distinctly higher than that of F intermediate, 785/750 cm(-1). The rate of reaction from P to F in D(2)O solution is evidently slower than that in H(2)O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H(2)O(2) gave the nu(Fe=O) band at 803/769 cm(-1) for H(2)(16)O(2)/H(2)(18)O(2) and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O(2) provided the P intermediate (P(M)) giving rise to an absorption peak at 607 nm and the nu(Fe=O) bands at 804/768 cm(-1). Thus, three kinds of P intermediates are considered to have the same oxo-heme a(3) structure. The nu(4) and nu(2) modes of heme a(3) of the P intermediate were identified at 1377 and 1591 cm(-1), respectively. The Raman excitation profiles of the nu(Fe=O) bands were different between P and F. These observations may mean the formation of a pi cation radical of porphyrin macrocycle in P.     

Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen

Located within the perisinusoidal space and surrounded by extracellular matrix, hepatic stellate cells (HSC) undergo phenotypic trans-differentiation called "myofibroblastic activation" in liver fibrogenesis. This study investigated the regulation of interleukin-1 (IL-1alpha) on expression of matrix metalloproteinases (MMPs) by HSC grown in three-dimensional extracellular matrix and the role of MMPs in HSC activation. To recapitulate the in vivo "quiescent" state of HSC, the isolated rat HSC were grown in three-dimensional Matrigel or type I collagen. Stimulation with IL-1alpha caused robust induction of pro-MMP-9 (the precursor of matrix metalloproteinase-9) when HSC were cultured in these matrices. IL-1alpha induced a conversion of the pro-MMP-9 to the active form only when the cells were in type I collagen. In collagen lattices, IL-1alpha provoked activation of HSC with induction of MMP-13, MMP-3, and breakdown of the matrix. The HSC activation was completely prevented by a treatment of the cells with tissue inhibitor of metalloproteinase-1 or deprivation of MMP-9. Once fully activated, HSC failed to express MMP-9 and showed attenuated induction of MMP-13 and MMP-3. Further, we demonstrated colocalization of alpha-smooth muscle actin and MMP-9 in a subpopulation of HSC in human fibrotic liver tissues. Thus, this study provides a novel model to enlighten the role of MMPs, particularly that of MMP-9, in HSC activation regulated by a specific cytokine in liver fibrogenesis.     

Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.     

Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase

Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.     

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.     

Identification and characterization of a protostome homologue of peropsin from a jumping spider

Peropsin, a member of the opsin family, has characteristics of two functionally distinct opsin-groups, that is, amino acid residues conserved among opsins for light-sensing and a retinal-photoisomerase-like molecular property. Although such a bilateral feature of peropsin seems to be important for understanding the diversity of the opsin family, previous studies have been limited to higher deuterostome, vertebrate and amphioxus peropsins. Here, we report a protostome peropsin homologue from a jumping spider. We found a spider opsin that shares amino acid homology and conserved amino acid residues with known peropsins. The spider opsin-based pigment heterologously expressed in cultured cells exhibited photoisomerase-like isomerization characteristics and a bistable nature. Based on the characteristics of both the amino acid homology and its photochemical properties, we concluded that the spider opsin is the first protostome peropsin homologue. These results show that peropsin existed before the deuterostome–protostome split like other members of the opsin family. In addition, the spider peropsin was localized to non-visual cells in the retina, and fluorescence from reduced retinal chromophore was also observed in the region where peropsin was localized. These findings provide the first demonstration that the peropsin can form a photosensitive pigment in vivo and underlie non-visual function.     

Moxibustion activates host defense against herpes simplex virus type I through augmentation of cytokine production

Moxibustion is a technique used in traditional oriental medicine, the aim of which is to cure and/or prevent illness by activating a person's ability for self‐healing. In this study, we assessed how moxibustion would affect the immune system and whether it would augment protective immunity. Mice were treated with moxibustion at Zusanli (ST36) acupoints; we analyzed mortality and cytokine activity in sera after infection with herpes simplex virus type 1 (HSV‐1), and cytokine gene expression in the skin and the spleen without a virus challenge. Our study demonstrates that pretreatment of BALB/c mice with moxibustion resulted in a marked increase in the survival rate after infection with lethal doses of HSV‐1, and elevated serum levels of IL‐1β and IFN‐γ on days 1 and 6 post‐infection with HSV‐1. Semi‐quantitative RT‐PCR assay showed that moxibustion treatment augmented the expression of IL‐1α, IL‐1β, IL‐6, universal‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the skin, and IL‐1α, IL‐1β, IL‐12p40, IL‐15, u‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the spleen. Moreover, moxibustion induces augmentation of natural killer cell activity. Collectively, our study demonstrates that moxibustion activates protective responses against HSV‐1 infection through the activation of cytokine production including IFN, and of NK cells.