Focused Proteomics Analysis of Habu Snake (Protobothrops flavoviridis) Venom Using Antivenom-Based Affinity Chromatography Reveals Novel Myonecrosis-Enhancing Activity of Thrombin-Like Serine Proteases.

Snakebites are one of the major causes of death and long-term disability in the developing countries due to the presence of various bioactive peptides and proteins in snake venom. In Japan, the venom of the habu snake (Protobothrops flavoviridis) causes severe permanent damage due to its myonecrotic toxins. Antivenom immunoglobulins are an effective therapy for snakebites, and antivenom was recently developed with effective suppressive activity against myonecrosis induced by snake venom. To compare the properties of an antivenom having anti-myonecrotic activity with those of conventional antivenom with no anti-myonecrotic activity, this study applied focused proteomics analysis of habu venom proteins using 2D gel electrophoresis. As a target protein for antivenom immunoglobulins with anti-myonecrotic activity, we identified a thrombin-like serine protease, TLSP2 (TLf2), which was an inactive proteolytic isoform due to the replacement of the active site, His43 with Arg. Additionally, we identified the unique properties and a novel synergistic function of pseudoenzyme TLf2 as a myonecrosis-enhancing factor. To our knowledge, this is the first report of a function of a catalytically inactive snake serine protease.

Diversified Biomineralization Roles of Pteria penguin Pearl Shell Lectins as Matrix Proteins.

Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related ß-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%-50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alone showed no effect on CaCO3 crystalization; however, PPL4 regulated the size of crystals collaborated with N-acetyl-D-glucosamine and chitin oligomer, which are specific in recognizing carbohydrates for PPL4. These observations highlight the unique functions and molecular evolution of this lectin family involved in the mollusk shell formation.

Glycan Binding Profiling of Jacalin-Related Lectins from the Pteria Penguin Pearl Shell.

We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + ß, ß + ß, respectively) and PPL4, which is heterodimer consisting of α + ß subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35-50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycans. On the other hand, PPL4 binds to high-mannose-and hybrid-type N-linked glycans but not agalactosylated- and galactosylated-type glycans.

Pufferfish Saxitoxin and Tetrodotoxin Binding Protein (PSTBP) Analogues in the Blood Plasma of the Pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca.

Pufferfish saxitoxin and tetrodotoxin (TTX) binding protein (PSTBP) is a glycoprotein that we previously isolated from the blood plasma of the pufferfish Takifugu pardalis; this protein was also detected in seven species of the genus Takifugu. We proposed that PSTBP is a carrier protein for TTX in pufferfish; however, PSTBP had not yet been found in genera other than Takifugu. In this study, we investigated the presence of PSTBP-like proteins in the toxic pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca. On the basis of ultrafiltration experiments, TTX was found to be present and partially bound to proteins in the plasma of these pufferfish, and Western blot analyses with anti-PSTBP antibody revealed one or two bands per species. The observed decreases in molecular mass following deglycosylation with glycopeptidase F suggest that these positive proteins are glycoproteins. The molecular masses of the deglycosylated proteins detected in the three Arothron species were larger than that of PSTBP in the genus Takifugu, whereas the two bands detected in C. patoca had molecular masses similar to that of tributyltin-binding protein-2 (TBT-bp2). The N-terminal amino acid sequences of 23⁻29 residues of these detected proteins were all homologous with those of PSTBP and TBT-bp2.

Isolation of Rice Bran Lectins and Characterization of Their Unique Behavior in Caco-2 Cells.

Rice bran lectins, named as RBA1 and RBA2, were isolated from Oryza sativa in two chromatography steps: affinity chromatography and cation-exchange chromatography. RBA1 was found to be composed of a covalently linked heterodimer of 20- and 12-kDa subunits, and RBA2 was a noncovalently linked dimer of 12-kDa subunits. Both RBA1 and RBA2 bound to desialylated complex glycoproteins such as fetuin, α1-acid glycoprotein, and transferrin, and agalactosylated complex glycoproteins such as agalacto fetuin, agalacto-α1-acid glycoprotein, and agalacto-transferrin, in addition to chitooligosacchrides. RBAs were heat stable up to 80 °C and stable at pH 4-10. RBA1 increased the transport of the fluorescent marker, rhodamine 123, which is known to be transported via the P-glycoprotein-mediated efflux pathway across human intestinal Caco-2 cell monolayers. Furthermore, RBA1 itself was transported to the basolateral side of the monolayers via an endocytotic pathway.

Outer Membrane Proteins Derived from Non-cyanobacterial Lineage Cover the Peptidoglycan of Cyanophora paradoxa Cyanelles and Serve as a Cyanelle Diffusion Channel.

The cyanelle is a primitive chloroplast that contains a peptidoglycan layer between its inner and outer membranes. Despite the fact that the envelope structure of the cyanelle is reminiscent of Gram-negative bacteria, the Cyanophora paradoxa genome appears to lack genes encoding homologs of putative peptidoglycan-associated outer membrane proteins and outer membrane channels. These are key components of Gram-negative bacterial membranes, maintaining structural stability and regulating permeability of outer membrane, respectively. Here, we discovered and characterized two dominant peptidoglycan-associated outer membrane proteins of the cyanelle (∼2 × 10(6) molecules per cyanelle). We named these proteins CppF and CppS (cyanelle peptidoglycan-associated proteins). They are homologous to each other and function as a diffusion channel that allows the permeation of compounds with Mr <1,000 as revealed by permeability measurements using proteoliposomes reconstituted with purified CppS and CppF. Unexpectedly, amino acid sequence analysis revealed no evolutionary linkage to cyanobacteria, showing only a moderate similarity to cell surface proteins of bacteria belonging to Planctomycetes phylum. Our findings suggest that the C. paradoxa cyanelle adopted non-cyanobacterial lineage proteins as its main outer membrane components, providing a physical link with the underlying peptidoglycan layer and functioning as a diffusion route for various small substances across the outer membrane.

A novel strategy to produce sweeter tomato fruits with high sugar contents by fruit-specific expression of a single bZIP transcription factor gene.

Enhancement of sugar content and sweetness is desirable in some vegetables and in almost all fruits; however, biotechnological methods to increase sugar content are limited. Here, a completely novel methodological approach is presented that produces sweeter tomato fruits but does not have any negative effects on plant growth. Sucrose-induced repression of translation (SIRT), which is mediated by upstream open reading frames (uORFs), was initially reported in Arabidopsis AtbZIP11, a class S basic region leucine zipper (bZIP) transcription factor gene. Here, two AtbZIP11 orthologous genes, SlbZIP1 and SlbZIP2, were identified in tomato (Solanum lycopersicum). SlbZIP1 and SlbZIP2 contained four and three uORFs, respectively, in the cDNA 5'-leader regions. The second uORFs from the 5' cDNA end were conserved and involved in SIRT. Tomato plants were transformed with binary vectors in which only the main open reading frames (ORFs) of SlbZIP1 and SlbZIP2, without the SIRT-responsive uORFs, were placed under the control of the fruit-specific E8 promoter. Growth and morphology of the resulting transgenic tomato plants were comparable to those of wild-type plants. Transgenic fruits were approximately 1.5-fold higher in sugar content (sucrose/glucose/fructose) than nontransgenic tomato fruits. In addition, the levels of several amino acids, such as asparagine and glutamine, were higher in transgenic fruits than in wild-type fruits. This was expected because SlbZIP transactivates the asparagine synthase and proline dehydrogenase genes. This 'sweetening' technology is broadly applicable to other plants that utilize sucrose as a major translocation sugar.

The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

Effect of chum salmon egg lectin on tight junctions in Caco-2 cell monolayers.

The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of ß-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized ß-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

Isolation and biochemical characterization of Apios tuber lectin.

Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 µg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport.

Novel matrix proteins of Pteria penguin pearl oyster shell nacre homologous to the jacalin-related ß-prism fold lectins.

Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of Pteria penguin oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of ß subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and in vitro CaCO3 crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of P. penguin nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of P. penguin mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation.

Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma.

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD-L1), known as an immunomodulatory ligand, has suppressive effects on CSC-related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274(low) and CD274(high) cells from each cell line, and xenografted them into immunodeficient NOD/scid/γcnull (NOG) mice. We found that the CD274(low) cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274(high) cells. Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC-related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Effects of food lectins on the transport system of human intestinal Caco-2 cell monolayers.

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20-40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.

Galectins in the abdominal cavity of the conger eel Conger myriaster participate in the cellular encapsulation of parasitic nematodes by host cells.

Congerin is a proto-type galectin distributed on the skin and mucosal epithelia of the upper digestive tract of the Japanese conger eel Conger myriaster. It has at least 2 isotypes, namely, congerin I and II, and plays a role in bio-defense at the body surface. In the current study, we identified both isotypes in the peritoneal fluid and peritoneal cells of C. myriaster by western blot and mass spectrometry (MS)/MS analysis. Cucullanus nematodes parasitize the abdominal cavity of C. myriaster, and immunohistochemical analyses demonstrated that congerins can bind to both the body surface of the encapsulated nematodes and the encapsulating cells. Furthermore, adhesion of the peritoneal cells to Sepharose particles was greatly accelerated when the microspheres were coated with congerin. Indeed, this effect was significantly hampered by the addition of lactose. These results indicate that congerin participates in the cellular encapsulation of the Cucullanus nematode via the induction of cellular adhesion to the parasites depending on lectin-glycoside recognition.

Allosteric regulation of the carbohydrate-binding ability of a novel conger eel galectin by D-mannoside.

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of D-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that D-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type ß-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.

Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation.

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

Diversified carbohydrate-binding lectins from marine resources.

Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families.