Habu Snake Research Articles

Supramolecular Hybrids of Proteins from Habu Snake Venom with Discrete [Pt(CN)4]2- Complex.

The venom of the Habu snake Protobothrops flavoviridis (P. flavoviridis) is known to contain a diverse array of proteins and peptides, with a notable presence of phospholipase A2 (PLA2) enzymes. These PLA2 enzymes have been extensively studied for their function and molecular evolution. Nevertheless, several aspects, such as the physical properties and the self-assembly mechanism of hierarchical structure from the nanoscale to the microscale with different chemical compounds, remain poorly understood. This study aims to fill this knowledge gap by investigating the behavior of enzyme components purified from P. flavoviridis venom in the presence of anionic [Pt(CN)4]2- complexes, which have the potential for soft metallophilic interactions and interesting optical properties. The purified PLA2 isozymes were diluted in Dulbecco's phosphate buffered saline (D-PBS (-)) and combined with the anionic metal complex, resulting in the formation of microstructures several micrometers in size, which further grew to form fibrous structures. This novel approach of combining PLA2 enzymes with discrete functional metal complexes opens up exciting possibilities for designing flexible and functional supramolecular and biomolecular hybrid systems in aqueous environments. These findings shed light on the potential applications of snake venom enzymes in nanotechnology and bioengineering.

Phylogeny-Related Variations in Venomics: A Test in a Subset of Habu Snakes (Protobothrops).

We conducted a comparative analysis to unveil the divergence among venoms from a subset of Old World habu snakes (Protobothrops) in terms of venomic profiles and toxicological and enzymatic activities. A total of 14 protein families were identified in the venoms from these habu snakes, and 11 of them were shared among these venoms. The venoms of five adult habu snakes were overwhelmingly dominated by SVMP (32.56 ± 13.94%), PLA2 (22.93 ± 9.26%), and SVSP (16.27 ± 4.79%), with a total abundance of over 65%, while the subadult P. mangshanensis had an extremely low abundance of PLA2 (1.23%) but a high abundance of CTL (51.47%), followed by SVMP (22.06%) and SVSP (10.90%). Apparent interspecific variations in lethality and enzymatic activities were also explored in habu snake venoms, but no variations in myotoxicity were found. Except for SVSP, the resemblance of the relatives within Protobothrops in other venom traits was estimated to deviate from Brownian motion evolution based on phylogenetic signals. A comparative analysis further validated that the degree of covariation between phylogeny and venom variation is evolutionarily labile and varies among clades of closely related snakes. Our findings indicate a high level of interspecific variation in the venom proteomes of habu snakes, both in the presence or absence and the relative abundance of venom protein families, and that these venoms might have evolved under a combination of adaptive and neutral mechanisms.

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.

TFPR1 acts as an immune regulator and an efficient adjuvant for proteins and peptides by activating immune cells, primarily through TLR2

Triflin, a non-toxic protein found in the venom of the Habu snake, belongs to the CRISP (cysteine-rich secretory protein) family, which comprises two domains: a C-terminal cysteine-rich domain (CRD) and an N-terminal pathogenesis-related-1 (PR-1) domain. The function of the highly structurally conserved PR-1 domain is unknown. Here, we successfully expressed the PR-1 domain of triflin (hereafter called TFPR1) in E. coli. Animal experiments showed that TFPR1 augmented Th1-biased antibody- and cell-mediated immune responses in mice immunized with two protein antigens (OVA and HBsAg) or a peptide antigen (HIV-1 pep). A flow cytometry-based binding assay and in vitro stimulation with TFPR1 showed that it triggered Th1-biased proinflammatory and immunoregulatory cytokine secretion primarily by binding to B cells and macrophages within the mouse splenocyte population. Quantitative RT-PCR, antibody blocking assays using a specific anti-mTLR2 antibody, and stimulatory experiments in vitro using splenocytes from TLR2-KO mice demonstrated that TFPR1 activated murine immune cells, primarily by stimulating toll-like receptor 2 (TLR2). These results suggest that TFPR1 acts as a novel immune modulator and potent adjuvant primarily by activating TLR2. Thus, the PR-1-based core domain might play a role in immune regulation.

Alternative mRNA Splicing in Three Venom Families Underlying a Possible Production of Divergent Venom Proteins of the Habu Snake, Protobothrops flavoviridis.

Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake.

Parathyroid Hormone–Related Protein Contributes to Early Healing of Habu Snake Venom–Induced Glomerulonephritis in Mice

Proliferative glomerulonephritis is characterized by local inflammation and mesangial cell deterioration, followed by mesangial proliferation and glomerular healing. Parathyroid hormone-related peptide (PTHrP) is a mesangial cytokine-like growth factor implicated in mesangial proliferation and survival. No data are available about its role in glomerulonephritis. Herein, we analyzed the expression and role of PTHrP in glomerular inflammation and healing in an experimental model of glomerulonephritis induced by i.v. injection of Habu snake venom in mice. The temporal analysis showed marked renal damage in the first days after venom injection and the beginning of recovery within 7 days. Glomerular expression of PTHrP (transcript and protein) was observed in the early phase after venom injection (from day 1 to day 3), along with an inflammatory environment. The inactivation of secreted PTHrP with PTHrP-neutralizing antibody (PTH2E11; 120 μg i.p. daily) reduced the markers of local inflammation (expression of macrophage chemotactic protein-1; regulated upon activation, normal T cell expressed and secreted; cyclooxygenase 2; IL-6; and macrophage infiltration) and abolished the expression of PTHrP itself. Moreover, the glomerular cell proliferation was hampered, and the healing process was prevented on day 7 after venom injection. These results show that PTHrP has antinomic actions in glomerulonephritis, participating in both the proinflammatory condition and the healing process. Our work reveals the essential role of PTHrP in early glomerular repair in an experimental model of glomerulonephritis.

Comparative Proteomics Analysis of Mouse Habu Nephritis Models with and without Unilateral Nephrectomy

Background/Aims: Individuals possessing a single kidney are at greater risk of renal injury upon exposure to harmful stimuli. This study aimed to explore the pathogenesis of renal injury in glomerulonephritis with versus without unilateral nephrectomy (UNX). Methods: Histological analysis and label-free quantitative proteomics were performed on two models—the Habu snake venom-induced glomerulonephritis model with versus without UNX (HabuU and Habu models, respectively). The role of villin 1, a differentially expressed protein (DEP) in mouse mesangial cells, was investigated. Results: Persistent mesangiolysis and focal hypercellularity together with reduced activation of cell proliferation in the HabuU model induced more serious renal injury compared with that in the Habu model. The DEPs between the two models were identified by label-free liquid chromatography-mass spectrometry. The KEGG pathway results indicated that regulation of actin cytoskeleton and focal adhesion were specifically enriched in the HabuU model. The cytoskeleton regulation protein villin 1 was downregulated in the HabuU model, but unchanged in the Habu model. Knockdown of villin 1 promoted apoptosis and inhibited the proliferation of mouse mesangial cells, suggesting villin 1 to be involved in qlomerular lesion self-repair insufficiency. Conclusion: By assessing the proteomic profiles of the two models, this study identified several important differences, particularly villin 1 expression, in regulatory mechanisms between the two models. Our findings provide novel insight into the mechanism of serious renal injury in glomerulonephritis with UNX.

Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity.Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content.Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column.Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible).Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. DisclosuresMercury:Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

Microcirculation-on-a-Chip: A Microfluidic Platform for Assaying Blood- and Lymphatic-Vessel Permeability.

We developed a microfluidic model of microcirculation containing both blood and lymphatic vessels for examining vascular permeability. The designed microfluidic device harbors upper and lower channels that are partly aligned and are separated by a porous membrane, and on this membrane, blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were cocultured back-to-back. At cell-cell junctions of both BECs and LECs, claudin-5 and VE-cadherin were detected. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules. Moreover, our results showed that the flow culture established in the device promoted the formation of endothelial cell-cell junctions, and that treatment with histamine, an inflammation-promoting substance, induced changes in the localization of tight and adherens junction-associated proteins and an increase in vascular permeability in the microdevice. These findings indicated that both BECs and LECs appeared to retain their functions in the microfluidic coculture platform. Using this microcirculation device, the vascular damage induced by habu snake venom was successfully assayed, and the assay time was reduced from 24 h to 30 min. This is the first report of a microcirculation model in which BECs and LECs were cocultured. Because the micromodel includes lymphatic vessels in addition to blood vessels, the model can be used to evaluate both vascular permeability and lymphatic return rate.

Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae).

Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

Enhanced vascular endothelial growth factor signaling in islets contributes to β cell injury and consequential diabetes in spontaneously diabetic Torii rats

AimsSpontaneously diabetic Torii (SDT) rats exhibit vascular abnormalities in pancreatic islets as the initial changes at pre-diabetes stage (8 weeks old), which is followed by β cell deterioration. In the present study, we investigated pathophysiological interactions between β cells and intra-islet microvasculature of SDT rats at pre- and peri-onset of diabetes. MethodsSDT rats were treated with Habu snake venom (HSV) to assess its hemorrhagic effects in glomeruli and pancreatic islets. SDT rats were treated with streptozotocin (STZ) to assess acute β cell fragility toward cytotoxic insult and the late-stage consequence of β cell ablation in neighboring structures. The receptor tyrosine kinase inhibitor sunitinib was administered to SDT rats to examine its therapeutic effect. ResultsHSV administration at 5 weeks old induced severe hemorrhage in and around islets in SDT rats. By contrast, precedent β cell depletion using STZ ameliorated hemorrhage, inflammation, and fibrosis around the islets at 13 weeks old, which is normally seen in SDT rats of this age. Blockade of vascular endothelial growth factor (VEGF)-like activity attenuated HSV-induced hemorrhage in SDT islets. VEGF release from SDT islets was increased at 13 weeks old but not at 5 weeks old, while interleukin-1β release was increased as early as 5 weeks old. Sunitinib treatment started at 5 weeks of age inhibited the onset of intra-islet hemorrhage, β cell loss, and hyperglycemia in SDT rats. ConclusionsEnhanced VEGF signaling in islets contributes to β cell injury, microvascular failure, and consequential diabetes in SDT rats.

Mesangial cell αvβ8-integrin regulates glomerular capillary integrity and repair.

αvβ8-Integrin is most abundantly expressed in the kidney, brain, and female reproductive organs, and its cognate ligand is latent transforming growth factor (LTGF)-β. Kidney αvβ8-integrin localizes to mesangial cells, and global β8-integrin gene (Itgb8) deletion results in embryonic lethality due to impaired placentation and cerebral hemorrhage. To circumvent the lethality and better define kidney αvβ8-integrin function, Cre-lox technology was used to generate mesangial-specific Itgb8-null mice. Platelet-derived growth factor-β receptor (PDGFBR)-Cre mice crossed with a reporter strain revealed functional Cre recombinase activity in a predicted mesangial pattern. However, mating between two different PDGFBR-Cre or Ren1(d)-Cre strains with Itgb8 (flox/-) mice consistently resulted in incomplete recombination, with no renal phenotype in mosaic offspring. Induction of a renal phenotype with Habu snake venom, a reversible mesangiolytic agent, caused exaggerated glomerular capillary microaneurysms and delayed recovery in Cre(+/-) PDGFRB (flox/-) mice compared with Cre(+/-) PDGFRB (flox/+) control mice. To establish the mechanism, in vitro experiments were conducted in Itgb8-null versus Itgb8-expressing mesangial cells and fibroblasts, which revealed β8-integrin-regulated adhesion to Arg-Gly-Asp (RGD) peptides within a mesangial-conditioned matrix as well as β8-integrin-dependent migration on RGD-containing LTGF-β or vitronectin matrices. We speculate that kidney αvβ8-integrin indirectly controls glomerular capillary integrity through mechanical tension generated by binding RGD peptides in the mesangial matrix, and healing after glomerular injury may be facilitated by mesangial cell migration, which is guided by transient β8-integrin interactions with RGD ligands.

Structural analysis and characterization of new small serum proteins from the serum of a venomous snake (Gloydius blomhoffii).

Some snakes have several anti-toxic proteins in their sera that neutralize their own venom. Five new small serum proteins (SSPs) were isolated from Japanese mamushi (Gloydius blomhoffii) serum by gel-filtration and RP-HPLC, and their N-Terminal sequences were determined. The amino acid sequences of the precursor proteins were deduced from the nucleotide sequences of cDNAs encoding them. Due to the sequence similarity to those of SSPs in habu snake (Protobothrops flavoviridis) serum (>75% identity), these proteins were designated mSSP-1 to mSSP-5 as the homologs of habu proteins. mSSP-1 was stable at 100 °C and in the pH range of 1-10, and inhibited the proteolytic activity of a certain snake venom metalloproteinase. The inhibitory activity was extinguished by modifying the amino groups of mSSP-1. mSSP-1 is the first prostate secretory protein of the 94 amino acid-family protein with a carbohydrate chain in the Asn37 residue.

Accelerated Evolution of Fetuin Family Proteins inProtobothrops flavoviridis(Habu Snake) Serum and the Discovery of an L1-Like Genomic Element in the Intronic Sequence of a Fetuin-Encoding Gene

Habu serum factor (HSF) and HSF-like protein (HLP) are fetuin family proteins isolated from Protobothrops flavoviridis (habu snake) serum with different physiological activities. A comparison of their cDNAs and intronic sequences revealed that nucleotide substitutions were primarily in protein-coding regions, and the substitution patterns indicated accelerated evolution of these proteins. Genomic DNA fragment analysis, including intron 1, revealed a 6.6-kb insertion homologous to the full-length mammalian LINE1 (L1) retrotransposable element (PfL1) only in the HLP gene. This segment retains an open reading frame (ORF) that encodes a reverse transcriptase (RT)-like protein (PfRT). We further found that a large number of homologous segments have dispersed in the habu snake genome, although we could not determine the enzymatic activities of their products. Moreover, an analysis of habu snake liver RNA indicated active transcription of the PfRT genes, suggesting that high levels of RT activity in this snake have driven the evolution of unique phenotypes of venom enzymes and serum inhibitors of them.

Glomerular Repair Retardation via Blocking of Angiotensin II Type 1a Receptor Pathway in a Mouse Glomerulonephritis Model

Background/Aims: To examine the role of the angiotensin II (ATII) type 1a receptor (AT1-R) pathway in renal tissue damage and repair, we investigated reversible glomerular injury in a mouse model of habu snake venom (HSV)-induced glomerulonephritis using AT1-R-deficient (AT1a-/-) mice and AT1-R antagonist-treated mice. Methods: Experimental glomerulonephritis was induced by single administration of HSV to AT1a+/+ mice (HSV group) and AT1a-/- mice (KO-HSV group) and AT1-R antagonist-treated BL6 mice (HSV-ARB group). Morphological change and expression levels of type IV collagen, CD31, and vascular endothelial growth factor (VEGF) were analyzed. Results: The HSV group showed increased mesangial matrix expansion on day 7, which returned to preinjection levels by day 56, while mes-angial matrix expansion and increased type IV collagen expression were seen throughout days 7 to 56 in the KO-HSV group. The KO-HSV group showed fewer CD31-positive capillary loops and a marked decrease in the number of VEGF-positive cells in the glomeruli than the HSV group. VEGF administration to the KO-HSV group facilitated glomerular capillary repair and reconstruction. The HSV-ARB group showed the same delay in glomerular repair as that seen in the KO-HSV group. Conclusion: Our results indicate that blocking of the ATII-AT1R pathway delays glomerular repair via angiogenesis inhibition, followed by reduced induction of VEGF.

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.

Production and degradation of extracellular matrix in reversible glomerular lesions in rat model of habu snake venom-induced glomerulonephritis

We investigated the mechanism of development and repair process of glomerular injury in a rat model of habu snake (Trimeresurus flavoviridis) venom (HSV)-induced glomerulonephritis. Glomerulonephritis was induced in rats by intravenously injecting HSV at 3 mg/kg. Renal tissue was isolated and subjected to immunohistochemical analysis for expression levels of type IV collagen, heat shock protein 47 (HSP47), transforming growth factor-β (TGF-β), and matrix metalloproteinase-3 (MMP-3), as well as its transcription factor Ets-1. Expression levels of HSP47, TGF-β, and type IV collagen began to increase in the mesangial area starting from day 14 and peaked on day 21, followed by a gradual decrease. Expression levels of MMP-3 and Ets-1 started to increase coinciding with peak production of mesangial matrix on day 21, peaking on day 35, followed by gradual decrease. Expression of MMP-3 and Ets-1 persisted until day 63, whereas that of HSP47 and type IV collagen returned to baseline level at this time point. Time-course changes of extracellular matrix (ECM) accumulation in glomeruli in the HSV-induced glomerulonephritis model were correlated with those of factors involved in both ECM production and degradation systems. Continued expression of factors in the degradation system seems particularly important for the repair process. These findings might lead to new therapies that prevent and repair glomerular injury.

Small serum protein-1 changes the susceptibility of an apoptosis-inducing metalloproteinase HV1 to a metalloproteinase inhibitor in habu snake (Trimeresurus flavoviridis)

Viperidae snakes containing various venomous proteins also have several anti-toxic proteins in their sera. However, the physiological function of serum protein has been elucidated incompletely. Small serum protein (SSP)-1 is a major component of the SSPs isolated from the serum of a Japanese viper, the habu snake (Trimeresurus flavoviridis). It exists in the blood as a binary complex with habu serum factor (HSF), a snake venom metalloproteinase inhibitor. Affinity chromatography of the venom on an SSP-1-immobilized column identified HV1, an apoptosis-inducing metalloproteinase, as the target protein of SSP-1. Biacore measurements revealed that SSP-1 was bound to HV1 with a dissociation constant of 8.2 × 10⁻⁸ M. However, SSP-1 did not inhibit the peptidase activity of HV1. Although HSF alone showed no inhibitory activity or binding affinity to HV1, the SSP-1-HSF binary complex bound to HV1 formed a ternary complex that non-competitively inhibited the peptidase activity of HV1 with a inhibition constant of 5.1 ± 1.3 × 10⁻⁹ M. The SSP-1-HSF complex also effectively suppressed the apoptosis of vascular endothelial cells and caspase 3 activation induced by HV1. Thus, SSP-1 is a unique protein that non-covalently attaches to HV1 and changes its susceptibility to HSF.

Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies

The vascular endothelial growth factor (VEGF) and its receptor (VEGFR) have been shown to play major roles not only in physiological but also in most pathological angiogenesis, such as cancer. VEGF belongs to the PDGF supergene family characterized by 8 conserved cysteines and functions as a homodimer structure. VEGF-A regulates angiogenesis and vascular permeability by activating 2 receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk1 in mice). On the other hand, VEGF-C/VEGF-D and their receptor, VEGFR-3 (Flt-4), mainly regulate lymphangiogenesis. The VEGF family includes other interesting variants, one of which is the virally encoded VEGF-E and another is specifically expressed in the venom of the habu snake (Trimeresurus flavoviridis). VEGFRs are distantly related to the PDGFR family; however, they are unique with respect to their structure and signaling system. Unlike members of the PDGFR family that strongly stimulate the PI3K-Akt pathway toward cell proliferation, VEGFR-2, the major signal transducer for angiogenesis, preferentially utilizes the PLCγ-PKC-MAPK pathway for signaling. The VEGF-VEGFR system is an important target for anti-angiogenic therapy in cancer and is also an attractive system for pro-angiogenic therapy in the treatment of neuronal degeneration and ischemic diseases.

Conditional VHL gene deletion activates a local NO-VEGF axis in a balanced manner reinforcing resistance to endothelium-targeted glomerulonephropathy

We have reported that tubular epithelial cell injury caused by renal ischemia-reperfusion is attenuated in conditional VHL knockout (VHL-KO) mice and also that induction of hypoxia-inducible factor (HIF) suppresses angiotensin II-accelerated Habu snake venom (HV) glomerulonephropathy in rats. However, it remains unknown whether VHL knockdown protects glomerular endothelial cells from endothelium-targeted glomerulonephritis. VHL-KO mice with HV glomerulonephropathy (HV GN) had fewer injured glomeruli, a lower mesangiolysis score and reduced blood urea nitrogen levels. Immunoreactivity of vascular endothelial growth factor (VEGF) in the glomerular capillaries was enhanced by VHL knockdown and was conserved even in VHL-KO mice with HV GN, despite HV-attenuating endothelial VEGF expression in vitro. VHL-KO mice showed enhanced nitric oxide (NO) production in glomerular endothelial cells and tubular cells, associated with activated VEGF expression in the kidney (i.e. an activated NO-VEGF axis). The levels of NO in glomeruli and tubules were conserved even in mice with HV GN. In contrast, suppressing NO production in glomerular endothelial cells by an NO synthase inhibitor, N(ϖ)-nitro-L-arginase, completely blunted the protection of VHL-KO from HV GN. The activated NO-VEGF axis in the kidney of VHL-KO mice was also associated with an elevation in Flk-1 phosphorylation and increased levels of IL-10 and IP-10. Conditional VHL knockdown may enhance the NO-VEGF axis and protect glomerular endothelial cells from HV GN, thereby providing resistance to injury of tubular epithelial cells and glomerular endothelial cells.