Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.     

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.     

Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: Molecular sequence analysis of its cDNA and biochemical properties

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.     

Proteomic analysis of the venom of the social wasp Apoica pallens (Hymenoptera: Vespidae)

Wasps are a diverse group of insects that possess a sting apparatus associated with a venom gland, which is used for predation and colony defense. The biochemistry of Hymenoptera venom has been evaluated in relation to allergy and immunology, and proteomics has been shown to be a powerful tool for the identification of compounds with pharmacological potential. Data on wasps venom the of genus Apoica are scarce, so the objective of the present work was to identify the venom proteins of the eusocial wasp Apoica pallens, as a first step towards further investigation of applied uses of the venom and its protein constituents. The venom proteins were separated by two-dimensional gel electrophoresis, followed by MALDI-TOF/TOF mass spectrometry. A total of 259 spots were detected, with molecular weights from 4.9 to 141 kDa. Thirty of these proteins were identified and classified into eight functional categories: allergen, enzyme, metabolism, structural, environmental response, proteoglycan, active in DNA and RNA, and unknown function. Due to the few available proteomic data for wasp venom, many proteins could not be identified, which makes studies with proteomic analysis of Hymenoptera venom even more important.     

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with 10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.     

Isolation,characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.)

Two novel lectins were isolated from roots and leaves of garlic. Characterization of the purified proteins indicated that the leaf lectin ASAL is a dimer of two identical subunits of 12 kDa, which closely resembles the leaf lectins from onion, leek and shallot with respect to its molecular structure and agglutination activity. In contrast, the root lectin ASARI, which is a dimer of subunits of 15 kDa, strongly differs from the leaf lectin with respect to its agglutination activity. cDNA cloning of the leaf and root lectins revealed that the deduced amino acid sequences of ASAL and ASARI are virtually identical. Since both lectins have identical N-terminal sequences the larger Mr of the ASARI subunits implies that the root lectin has an extra sequence at its C-terminus. These results not only demonstrate that virtually identical precursor polypeptides are differently processed at their C-terminus in roots and leaves but also indicate that differential processing yields mature lectins with strongly different biological activities. Further screening of the cDNA library for garlic roots also yielded a cDNA clone encoding a protein composed of two tandemly arrayed lectin domains. Since the presumed two-domain root lectin has not been isolated yet, its possible relationship to the previously described two-domain bulb lectin could not be studied at the protein level.     

Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja)

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.     

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.     

Factor X activator from Vipera lebetina snake venom,molecular characterization and substrate specificity

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6–9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly – factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly – factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg⁵²-Ile⁵³ bond in the heavy chain of human factor X and the Arg²²⁶-Val²²⁷ bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.     

Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom

The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.     

Purification,properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.     

Biochemical and molecular characterization and expression of the 11S-type storage protein from Coffea arabica endosperm

In order to define better the endosperm protein content of commercial coffee species Coffea arabica (Arabica) and C. canephora (Robusta), the principal storage protein of coffee grains has been analysed by 2-dimensional electrophoresis (2DE) and amino acid microsequencing. The most abundant polypeptide spots observed on mature coffee grain 2DE profiles were found to be subunits of the same protein, which exists as multiple isoforms with varying pIs. Strong sequence similaritywas found to the 11S family of plant storage proteins. The structure is typical of the 11S type, which occurs as a precursor of 55 kDa, and is observed under denaturing and reducing conditions on 2DE profiles in the form of cleavage products at approximately 20 kDa (β arms) and 32 kDa (α arms). Differences between Arabica and Robusta 2DE profiles indicate a secondary 11S protein family in some varieties of the latter. The existence of multiple pI forms may indicate that a multigene family encodes for these proteins. We estimate that the protein accounts for approximately 45 % of total grain protein. A cloned full-length cDNA of 1 706 bp coding for one of the isoforms is described and discussed in relation to other coffee storage protein sequences.     

Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity

Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has K_m 7.6 μM and V_max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.