Requirement of Ca(2+) Ions for the Hyperthermostability of Tk-Subtilisin from Thermococcus kodakarensis

Tk-subtilisin, a hyperthermostable subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca(2+) ions. Four of them (Ca2-Ca5) are responsible for folding of Tk-subtilisin. In this study, to clarify the role of the other three Ca(2+) ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/ΔCa1 was determined to be 50% of that of Tk-subtilisin using the refolded protein. This result suggests that the Ca1 ion is required for the maximal activity of Tk-subtilisin. The refolding rates of all Pro-S324A derivatives were comparable to that of Pro-S324A (active site mutant of Pro-TKS), indicating that these Ca(2+) ions are not needed for folding of Tk-subtilisin. The stabilities of Pro-S324A/ΔCa1 and Pro-S324A/ΔCa6 were decreased by 26.6 and 11.7 °C, respectively, in T(m) compared to that of Pro-S324A. The half-lives of Tk-subtilisin/ΔCa6 and Tk-subtilisin/ΔCa7 at 95 °C were 8- and 4-fold lower than that of Tk-subtilisin, respectively. These results suggest that the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin.     

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A novel lectin structure was found for a 17-kDa α-d-galactose-binding lectin (termed “MytiLec”) isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45–52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1–4Galβ1–4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.     

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.     

Identification of Amino Acid Residues Required for the Substrate Specificity of Human and Mouse Chondroitin Sulfate Hydrolase (Conventional Hyaluronidase-4)

Human hyaluronidase-4 (hHYAL4), a member of the hyaluronidase family, has no hyaluronidase activity, but is a chondroitin sulfate (CS)-specific endo-β-N-acetylgalactosaminidase. The expression of hHYAL4 is not ubiquitous but restricted to placenta, skeletal muscle, and testis, suggesting that hHYAL4 is not involved in the systemic catabolism of CS, but rather has specific functions in particular organs or tissues. To elucidate the function of hyaluronidase-4 in vivo, mouse hyaluronidase-4 (mHyal4) was characterized. mHyal4 was also demonstrated to be a CS-specific endo-β-N-acetylgalactosaminidase. However, mHyal4 and hHYAL4 differed in the sulfate groups they recognized. Although hHYAL4 strongly preferred GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-containing sequences typical in CS-D, where GlcUA represents d-glucuronic acid, mHyal4 depolymerized various CS isoforms to a similar extent, suggesting broad substrate specificity. To identify the amino acid residues responsible for this difference, a series of human/mouse HYAL4 chimeric proteins and HYAL4 point mutants were generated, and their preference for substrates was investigated. A combination of the amino acid residues at 261–265 and glutamine at 305 was demonstrated to be essential for the enzymatic activity as well as substrate specificity of mHyal4.     

Variation in Courtship Ultrasounds of Three Ostrinia Moths with Different Sex Pheromones

Moths use ultrasounds as well as pheromones for sexual communication. In closely related moth species, variations in ultrasounds and pheromones are likely to profoundly affect mate recognition, reproductive isolation, and speciation. The European corn borer, Ostrinia nubilalis, and its Asian congeners, Ostrinia furnacalis and Ostrinia scapulalis, exhibit within-species and between-species variation in their pheromone communication. Recently, we reported ultrasound communication in O. furnacalis; however, variations in ultrasounds in the three congeners have not been addressed to date. Here we investigated features of ultrasound production and hearing in O. nubilalis and O. scapulalis, and compared them with those of O. furnacalis. As in O. furnacalis, males of O. nubilalis and O. scapulalis produced ultrasounds during courtship by rubbing specialized scales on the wings against scales on the thorax. The covering of these scales with nail polish muffled the sounds and significantly reduced mating success in O. nubilalis, showing the importance of ultrasound signaling in mating. The ultrasounds produced by O. nubilalis and O. scapulalis were similar, consisting of long trains of pairs of pulses with a main energy at 40 kHz, but distinctly different from the ultrasound produced by O. furnacalis, consisting of groups of pulses peaking at 50 kHz and with substantially more energy up to 80 kHz. Despite overall similarities, temporal features and patterns of amplitude modulation differed significantly among the geographic populations of O. nubilalis and O. scapulalis, which differed in pheromone type. In contrast, no significant difference in hearing was found among the three species with regard to the most sensitive frequencies and hearing threshold levels. The patterns of variations in the songs and pheromones well reflected those of the phylogenetic relationships, implying that ultrasound and pheromone communications have diverged concordantly. Our results suggest that concordant evolution in sexual signals such as courtship ultrasounds and sex pheromones occurs in moths.     

Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 at Ser34 Enhances Platelet-activating Factor Production in Endotoxin-stimulated Macrophages

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser³⁴ of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.