Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range

Two glucoamylase-like genes, TVN1315 and Ta0286, from the archaea Thermoplasma volcanium and T. acidophilum, respectively, were expressed in Escherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)₆ barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display K_m values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (K_i) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.     

Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase

Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca(2+). However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.     

Cryptic genetic divergence and associated morphological differentiation in the arboreal land snail Satsuma (Luchuhadra) largillierti (Camaenidae) endemic to the Ryukyu Archipelago, Japan

Land snails are renowned model organisms in evolutionary ecology, but extensive morphological variation and lack of readily available diagnostic characters often invite taxonomic confusion among closely related species. Satsuma (Luchuhadra) largillierti is an arboreal land snail endemic to Okinawa Island, Japan, in which extensive geographic variation in shell morphology has long caused taxonomic complication. To establish robust species limits among S. largillierti and its allies, we perform molecular and morphological analyses of snails sampled from the entire range of Luchuhadra. Analyses of the mitochondrial cytochrome oxidase subunit 1 gene and nuclear ribosomal internal transcribed spacer sequences divide S. largillierti into two, reproductively isolated groups. Each of these groups includes Satsuma erabuensis and Satsuma sooi, respectively, which occur on a nearby island and were traditionally regarded as distinct species. Morphometric analysis of the genitalia further provides clear difference between the two groups, whereas differentiation in shell morphology is slight if any. These results indicate that the traditional taxonomy has overly depended on inadequate morphological difference and a priori information of island geography, which resulted in oversimplification of complex speciation history in Luchuhadra.     

Highly multilayered urease decomposes highly concentrated urea

Urease was immobilized at a density of 1.2 g of urease per gram of a matrix via ion-exchange binding of urease to an anion-exchange polymer chain grafted onto a pore surface of a porous hollow-fiber membrane and subsequent cross-linking of urease with transglutaminase. Urea was hydrolyzed during the permeation of a urea solution, the concentration of which ranged from 2 to 8 M, through the pores of the resultant membrane with a thickness of approximately 1 mm. Quantitative hydrolysis of 4 M urea was achieved at a permeation rate lower than 1 mL/h, i.e., a residence time longer than 5.1 min, at ambient temperature. This performance is ascribed to convective transport of urea through the pores rimmed by the urease-immobilized polymer chains at a high density. Urease was denatured in the presence of urea at concentrations higher than 6 M while hydrolyzing urea.     

Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland,Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2^d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.     

Studies on conformational transitions of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. II. Conformational characteristics of stabilized reaction intermediates as revealed by fluorescent and paramagnetic probes

Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.     

The Semaphorin 3A Inhibitor SM-345431 Accelerates Peripheral Nerve Regeneration and Sensitivity in a Murine Corneal Transplantation Model

Background

Peripheral nerve damage of the cornea is a complication following surgery or infection which may lead to decreased visual function. We examined the efficacy of the semaphorin 3A inhibitor, SM-345431, in promoting regeneration of peripheral nerves in a mouse corneal transplantation model.

Methodology/Principal Findings

P0-Cre/Floxed-EGFP mice which express EGFP in peripheral nerves cells were used as recipients of corneal transplantation with syngeneic wild-type mouse cornea donors. SM-345431 was administered subconjunctivally every 2 days while control mice received vehicle only. Mice were followed for 3 weeks and the length of regenerating nerves was measured by EGFP fluorescence and immunohistochemistry against βIII tubulin. Cornea sensitivity was also measured by the Cochet-Bonnet esthesiometer. CD31 staining was used to determine corneal neovascularization as a possible side effect of SM-345431. Regeneration of βIII tubulin positive peripheral nerves was significantly higher in SM-345431 treated mice compared to control. Furthermore, corneal sensitivity significantly improved in the SM-345431 group by 3 weeks after transplantation. Neovascularization was limited to the peripheral cornea with no difference between SM-345431 group and control.

Conclusions/Significance

Subconjunctival injections of SM-345431 promoted a robust network of regenerating nerves as well as functional recovery of corneal sensation in a mouse keratoplasty model, suggesting a novel therapeutic strategy for treating neurotrophic corneal disease.     

Accumulation of Secretory Vesicles in the Lacrimal Gland Epithelia Is Related to Non-Sjögren's Type Dry Eye in Visual Display Terminal Users

Previous observations in a rat model of a non-Sjögren''s syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.     

Independent flexible motion of submolecular domains of the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum measured by time-resolved fluorescence depolarization of site-specifically attached probes

The Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was site-specifically labeled with either N-(1-anilinonaphth-4-yl)maleimide (ANM) or 5-[[(iodoacetamido)-ethyl]amino]naphthalene-1-sulfonate (IAEDANS), and the segmental motion of submolecular domains of the ATPase molecule was examined by means of time-resolved and steady-state fluorescence anisotropy measurements. The ANM-binding domain showed wobbling with a rotational relaxation time phi = 69 ns in the absence of free Ca2+ without any independent wobbling of the ANM moiety. The IAEDANS-binding domain showed a significantly slower wobbling with phi = 190 ns in the absence of Ca2+. The present results demonstrated for the first time that the ATPase molecule is composed of distinct domains whose mobilities are considerably different from each other. The binding of Ca2+ to the transport site increased the segmental motion of ANM-labeled domain, leading to a phi value of 65 ns. Solubilization of the ANM-labeled SR membranes by deoxycholate led to a further increase in the segmental flexibility (phi = 48 ns in the absence of free Ca2+), indicating that the mobility of the ANM-binding domain was considerably restricted through interaction with the membrane. The mobility of the ANM-binding domain of solubilized ATPase was also increased to some extent upon binding of Ca2+.