Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

When spider and snake get along: Fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell.

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies Against Loxosceles Spider Venom.

Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen's formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure.

Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause a massive inflammatory response, dermonecrosis, hemolysis and platelet aggregation, among other effects. Even though the PLDs induce strong platelet aggregation, there are no studies showing how the PLDs interact with platelets to promote this effect. Since many agonists must interact with specific receptors on the platelet membrane to induce aggregation, it is reasonable to expect that the PLDs may, in some way, also interact with platelets, to induce this activity. Therefore, to address this possibility, in this work, a recombinant PLD, called LgRec1, from L. gaucho was fused to enhanced green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 to platelets, by fluorescence-activated cell sorter (FACS) and confocal microscopy. The preservation of biological activities of this chimera toxin was also analyzed. As a first, the results show that LgRec1 does not require plasma components to bind to platelets, although these components are necessary to LgRec1 to induce platelet aggregation. Also, the attachment of LgRec1 to human platelets' cell membranes suggests that the exposure of phosphatidylserine (PS) may act as a scaffold for coagulation factors. Therefore, the results add new information about the binding of Loxosceles PLDs to platelets, which may help unravel how these toxins promote platelet aggregation.

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

Erratum to: A novel ELISA for diagnosis of Glanzmann's thrombasthenia and the heterozygote carriers.

Erratum to: Annals of Hematology 91(6): 917­921. DOI 10.1007/s00277-011-1390-1 . The authors inadvertently omitted 2 fellow authors from the author list: Dr. Diego Butera should be listed as the fourth author. His affiliation is Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, NSW, Australia. His contributions are as follows: Designed, synthesized and produced EcAPv. He has no competing interests to declare. Dr. Geraldo S. Magalhaes should be listed as the fifth author. His affiliation is Laboratory of Immunopathology, Butantan Institute, São Paulo, SP, Brazil. His contributions are as follows: Produced more EcAPv when requested in October 2009. He has no competing interests to declare.

Unraveling the processing and activation of snake venom metalloproteinases.

Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes responsible for most symptoms of human envenoming. Like matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAM) proteins, SVMPs are synthesized as zymogens, and enzyme activation is regulated by hydrolysis of their prodomain, but the processing of SVMPs is still unclear. In this study, we attempted to identify the presence of prodomain in different compartments of snake venom glands as zymogens or in the free form to elucidate some mechanism involved in SVMP activation. Using antibodies obtained by immunization with a recombinant prodomain, bands of zymogen molecular mass and prodomain peptides were detected mostly in gland extracts all along the venom production cycle and in the venom collected from the lumen at the peak of venom production. Prodomain was detected in secretory cells mostly in the secretory vesicles near the Golgi. We hypothesize that the processing of SVMPs starts within secretory vesicles and continues in the lumen of the venom gland just after enzyme secretion and involves different steps compared to ADAMs and MMPs but can be used as a model for studying the relevance of peptides resulting from prodomain processing and degradation for controlling the activity of metalloproteinases.

Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland.

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (~65%) and dermonecrosis (~100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.

Gene expression of inflammatory mediators induced by jararhagin on endothelial cells.

Snake venom metalloproteinases (SVMP) are abundant toxins in venoms of viper snakes and play a relevant role in the complex and multifactorial tissue damage characteristic of Viperidae envenoming. Jararhagin, a SVMP isolated from Bothrops jararaca venom, induces a fast onset hemorrhagic lesions acting directly on the capillary vessels, which are disrupted by toxin adhesion and degradation of extracellular matrix proteins like collagen IV. Jararhagin also triggers inflammatory response, where endothelial cells are activated, resulting in the enhanced rolling of circulating leukocytes, nitric oxide generation, prostacyclin production and pro-inflammatory cytokines release. Jararhagin also decreases endothelial cells viability inducing apoptosis (in vitro studies). In the present study we attempted to correlate the effect of sub-apoptotic doses of jararhagin on human umbilical vein endothelial cells (HUVECs) and gene expression of pro-inflammatory mediators, using microarray assay, real time PCR and detection of specific proteins on HUVEC surface or released in the medium. Jararhagin was effective in activate and up-regulate the gene expression of different mediators such as E-selectin, VCAM-1, IL-8, CD69, Ang-2 and MMP-10. Despite the increase in expression of genes coding for such molecules, jararhagin did not induce increased concentrations of E-selectin, VCAM-1 and IL-8 produced or released by endothelial cells. In conclusion, jararhagin is able to activate pro-inflammatory gene transcription on endothelial cells however this stimulus is not sufficient to result in the consequent expression of pro-inflammatory effectors molecules like E-selectin, VCAM-1 and IL-8. The time courses of these events, as well as the doses of jararhagin are important points to be addressed herein.

A novel ELISA for diagnosis of Glanzmann's thrombasthenia and the heterozygote carriers.

A sensitive and specific sandwich ELISA was developed for the diagnosis of Glanzmann's thrombasthenia (GT) and the heterozygote carriers of the disease using whole blood platelets. The assay used anti-CD36 antibody to capture platelets from platelet-rich plasma which was subsequently treated with a bioengineered disintegrin/alkaline phosphatase hybrid protein specific for GP IIb/IIIa. The test allows large number of samples to be typed and can also be used on stored samples. The assay correctly diagnosed 40 normal healthy individuals, 10 GT cases, 10 heterozygotes, 3 Bernard-Soulier syndrome cases and 2 type 3 GT cases. ELISA plates were stable at room temperature up to 3 weeks without any loss of activity. This novel and simple test can be widely used for heterozygote detection besides diagnosing GT cases without using a sophisticated flow cytometer or a platelet aggregometer and has wide applicability in countries like India where many of these cases remain undiagnosed due to the lack of diagnostic facilities.

Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs.

BACKGROUND: Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications. RESULTS: Thirty-one SVMP cDNAs were full length cloned from a single specimen of Bothrops neuwiedi snake, sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and class P-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. In opposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregation of class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their 5' ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIx sequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with other class P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains. Complementary regions within cDNA sequences were noted and may participate in recombination either at DNA or RNA levels. Proteins predicted by these cDNAs show the main features of the correspondent classes of SVMP, but P-IIb and P-IIx included two additional cysteines cysteines at the C-termini of the disintegrin domains in positions not yet described. CONCLUSIONS: In B. neuwiedi venom gland, class P-II SVMPs were represented by three different types of transcripts that may have arisen by interclass recombination with P-I and P-III sequences after the divergence of the different classes of SVMPs. Our observations indicate that exon shuffling or post-transcriptional mechanisms may be driving these recombinations generating new functional possibilities for this complex group of snake toxins.

Insularin, a disintegrin from Bothrops insularis venom: Inhibition ofplatelet aggregation and endothelial cell adhesion by the native andrecombinant GST-insularin proteins

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase highperformance liquid chromatography using a C18 column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, ncluding the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein withglutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADPinduced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

"Insularin, a disintegrin from Bothrops insularis venom: inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins".

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase high-performance liquid chromatography using a C(18) column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs

Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatiletoxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clottingproteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to thestructural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains,which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 ofthem belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and posttranslationalmodifications. Thirty-one SVMP cDNAs were full length cloned from a single specimen of Bothrops neuwiedi snake,sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and classP-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. Inopposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregationof class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their5’ ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIxsequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with otherclass P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains.Complementary regions within cDNA sequences were noted and may participate in recombination either at DNAor RNA levels.

Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to alpha2beta1 integrin and collagen.

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

Different regions of the class P-III snake venom metalloproteinasejararhagin are involved in binding to a2b1 integrin and collagen

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported thatjararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to a2b1 integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to a2b1 integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344–421), showed a significant binding to recombinant a2b1 integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding ofjararhagin to collagen, but not to recombinant a2b1 integrin nor to cell-surface-exposed a2b1 integrin (a2-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and a2b1 integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

A novel human G protein-coupled receptor is over-expressed in prostate cancer.

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological functions. The number of known members that belong to this large family of receptors has been rapidly increasing. Now, with the availability of the human genome sequence databases, further family members are being identified. We describe the identification of a novel GPCR that shows no significant amino acid identity to any one of the known members of the GPCR superfamily. The gene expression pattern of this receptor is restricted: in normal tissues it is confined to the nervous system and testis, but we also detected gene expression in several tumor types, most notably prostate cancer, suggesting a potential role for this gene as a marker for this disease.

A novel human G protein-coupled receptor is over-expressed in prostate cancer

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological functions. The number of known members that belong to this large family of receptors has been rapidly increasing. Now, with the availability of the human genome sequence databases, further family members are being identified. We describe the identification of a novel GPCR that shows no significant amino acid identity to any one of the known members of the GPCR superfamily. The gene expression pattern of this receptor is restricted: in normal tissues it is confined to the nervous system and testis, but we also detected gene expression in several tumor types, most notably prostate cancer, suggesting a potential role for this gene as a marker for this disease.