Agaritine derived from Agaricus blazei Murrill induces apoptosis via mitochondrial membrane depolarization in hematological tumor cell lines.

Objectives: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. Methods: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 µM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). Results: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. Conclusions: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

Quinonoid dihydropteridine reductase, a tetrahydrobiopterin-recycling enzyme, contributes to 5-hydroxytryptamine-associated platelet aggregation in mice.

Quinonoid dihydropteridine reductase (QDPR) regenerates tetrahydrobiopterin (BH4), which is an essential cofactor for catecholamine and serotonin (5-hydroxytryptamine, 5-HT) biosynthesis. Serotonin is known as an important platelet agonist, but its role under BH4-synthesizing or recycling enzymes deficiency is unknown. In the present study, we evaluated the effect of Qdpr gene disruption on platelet aggregation using knockout (Qdpr-/-) mice. Platelet aggregation was monitored by light transmission aggregometry using adenosine diphosphate (ADP) and collagen as agonists. We also assessed how platelet aggregation was modified by 5-HT recovery through supplementation with 5-hydroxytryptophan (5-HTP), a 5-HT precursor, or by blocking the serotonin 5-HT2A receptor. Platelet aggregation in the Qdpr-/- mice was significantly suppressed in comparison with that in wild-type (Qdpr+/+) mice, particularly at the maintenance phase of aggregation. 5-HT storage was decreased in Qdpr-/- platelets, and 5-HTP supplementation recovered not only the intraplatelet 5-HT levels but also platelet aggregation. In addition, 5-HT signal blockade using sarpogrelate suppressed platelet aggregation in Qdpr+/+ mice, and platelets in Qdpr-/- mice were hardly affected. Our results indicate that QDPR deficiency suppresses platelet aggregation by impairing 5-HT biosynthesis in mice.

Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans, Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and ß share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3ß were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and ß-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab.

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the United States. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggested that TAGX-0004 possessed better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

Blood group antigen A on von Willebrand factor is more protective against ADAMTS13 cleavage than antigens B and H.

BACKGROUND: ADAMTS13 specifically cleaves the peptide bond between Y1605 and M1606 within the VWF-A2 domain. OBJECTIVE: The VWF contains ABO(H) blood group antigens, which may influence the susceptibility of VWF to ADAMTS13. METHODS: Using a unique monoclonal antibody recognizing the Y1605 residue, we have developed a sandwich ELISA to analyze the generation of a VWF-DP by ADAMTS13 quantitatively. RESULTS: Production of VWF-DP after exposure to four different degrees of high shear stress was validated in comparison to the reduction in high-molecular-weight multimers using VWF multimer analysis. In analysis of plasma from 259 healthy individuals, plasma levels of VWF antigen (VWF:Ag) were significantly lower in blood group O than in the other groups and were significantly correlated with plasma VWF-DP levels. The ratio between VWF-DP and VWF:Ag was significantly higher in blood group O than in blood groups A and AB. The ratio in blood group B was also significantly higher than those in A and AB, but did not differ from blood group O. Finally, to examine whether ABO(H) blood group antigens contributed to VWF cleavage, 82 plasma samples were exposed to high shear stress using a cone-plate shear stress inducer. The difference in the VWF-DP/VWF:Ag ratio before and after high shear stress in blood group O was significantly greater than those in groups A and AB. CONCLUSION: These results indicate that blood group antigen A on VWF was more protective against ADAMTS13 cleavage than antigens B and H.

Effects of plasma glycosyltransferase on the ABO(H) blood group antigens of human von Willebrand factor.

Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

von Willebrand factor and von Willebrand disease.

von Willebrand factor (VWF) has two major roles in hemostasis, as a form of molecular glue which functions in platelet plug formation and as a protective transporter for coagulation factor VIII (FVIII). VWF shows a multimeric chain structure composed of 270 kDa subunits containing binding domains for FVIII, platelet and collagens. Biosynthesis, storage, secretion of VWF and the cleavage process by ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 repeats 13) regulating VWF activity have been elucidated. von Willebrand disease (VWD) is an autosomal bleeding disorder, first documented in 1926, caused by quantitative or qualitative deficiency of VWF. The classification and molecular pathogenesis of VWD have been developed during a 90-year period based on clinical laboratory analysis of VWF, and structure-function analysis of mutant VWF with amino acid substitutions (genetic changes). VWF is a unique and very large multifunctional plasma protein, the hemostatic activity of which is dynamically regulated by physiological shear stress in the blood stream.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galß1-4Glc). MytiLec revealed ß-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

A galactose-binding lectin isolated from Aplysia kurodai (sea hare) eggs inhibits streptolysin-induced hemolysis.

A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes α-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of d-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth.

Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα.

Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine.

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; Galα1-4Galß1-4Glc), 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.

Cytotoxicity and glycan-binding properties of an 18 kDa lectin isolated from the marine sponge Halichondria okadai.

A divalent cation-independent lectin-HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAcß1-4GlcNAcß1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4-12 and temperatures lower than 60 °C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner.

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination.

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the ß subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the ß subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the ß subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspß70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the ß subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

Cytotoxicity and glycan-binding profile of a D-galactose-binding lectin from the eggs of a Japanese sea hare (Aplysia kurodai).

A divalent cation-independent 16 kDa D-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized D-galactose and D-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl D-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galß1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k(ass) and k(diss) values are 2.4 × 10³ M⁻¹ s⁻¹ and 3.8 × 10⁻³ s⁻¹, respectively. AKL-2 appeared cytotoxicity against both Burkitt's lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937.

BACKGROUND: Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine. METHODS: Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells. RESULTS: Nuclear damage, DNA fragmentation, was observed by Wright-Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10µg/mL of agaritine for 48h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment. CONCLUSIONS: These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria. GENERAL SIGNIFICANCE: This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea).

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galß1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galß1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes ß1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.

Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody.

ADAMTS13 is a metalloproteinase that specifically cleaves unusually large von Willbrand factor multimers under high-shear stress. Deficiency of ADAMTS13 activity induces a life-threatening generalized disease, thrombotic thrombocytopenic purpura. We established a simple and efficient method to purify plasma ADAMTS13 (pADAMTS13) from cryosupernatant using an anti-ADAMTS13 monoclonal antibody (A10) that recognizes a conformational epitope within the disintegrin-like domain. Using the purified pADAMTS13, the amino acid residues involved in cleavage by thrombin, plasmin and leucocyte elastase were determined, and the carbohydrate moieties of this enzyme was analysed by lectin blots. Purified pADAMTS13 had a specific activity of 300 U/mg (25,057-fold purification) and the pI was 5.1-5.5. Cleavage sites of the purified pADAMTS13 by three proteases were identified; thrombin cleaved the four peptidyl bonds between Arg257-Ala258, Arg459-Ser460, Arg888-Thr889 and Arg1176-Arg1177, plasmin cleaved the three peptidyl bonds between Arg257-Ala258, Arg888-Thr889 and Arg1176-Arg1177, and elastase cleaved the two peptidyl bonds between Ile380-Ala381 and Thr874-Ser875. Lectin blot analysis indicated the presence of non-reducing terminal α2-6 and α2-3-linked sialic acid residues with penultimate ß-galactose residues on the N- and O-linked sugar chains of pADAMTS13, suggesting that pADAMTS13 is cleared from the circulation via the hepatic asialoglycoprotein receptor like other plasma glycoproteins.