L-amino acid oxidase from snake venom: Biotransformation and induction of apoptosis in human colon cancer cells.

This study evaluated the potential of antitumor activity of snake venom from Vipera ammodytes and L-amino acid oxidase from Crotalus adamanteus on different colorectal cancer cell lines through determination of cytotoxic activity by MTT assay, pro-apoptotic activity by acridine orange/ethidium bromide staining, and concentrations of redox status parameters (superoxide, reduced glutathione, lipid peroxidation) by colorimetric methods. The expression of genes involved in the biotransformation process and metabolite efflux was determined by qPCR method, while protein expression of glutathione synthetase and P-glycoprotein were analysed by immunocytochemistry. The analysis of cell death shows that snake venom dominantly leads cells to necrosis. Induction of apoptosis by L-amino acid oxidase was in correlation with oxidative disbalance in cancer cells. Gene expression profile of membrane transporters and CYP genes were different in each cell line and in correlation with their sensitivity of treatment. Our results show that L-amino acid oxidase from snake venom is a potent cytotoxic substance with pronounced pro-apoptotic activity. The inhibition of P-glycoprotein suggests that L-amino acid oxidase is a good substance for furter research of antitumor effect, with unexpressed potential for occurrence of drug resistance in vitro.

Identification and characterization of l-amino acid oxidase 2 gene in orange-spotted grouper (Epinephelus coioides).

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.

Cabbage defense response provoked by Trichoderma Th-LAAO.

To investigate the molecular mechanism of Trichoderma L-amino acid oxidase (Th-LAAO) in protecting and in promoting growth of cabbage infected with Botrytis cinerea, a three-way interaction system was established. Cabbage leaves treated with purified Th-LAAO significantly constrained damaged leaf area caused by B. cinerea infection. In response to Th-LAAO treatment, the expression levels of genes involved in photosynthesis, such as ribulose-1,5-bisphosphate carboxylase oxygenase, Rubisco activase, and ATP synthase increased 2.54, 2.18, and 1.41 folds, respectively. The transcription levels of sucrose transport protein 1 increased 7.6 fold. As to the expression of defense-related genes, the transcription level of ascorbate peroxidase increased 1.46 fold. On the contrary, pathogenesis-related protein 1, chitinase, ß-1,3 glucanase, and glutathione S-transferase decreased significantly. Overall, the results indicated that Th-LAAO may stimulate CO2 fixation and sucrose transport and elicit host defense responses in cabbage against B. cinerea, and this elicitation of defense response is likely to contribute to induced systemic resistance of host plant.

Prediction of novel inhibitors for Crotalus adamanteus l-amino acid oxidase by repurposing FDA-approved drugs: a virtual screening and molecular dynamics simulation investigation.

One of the deadliest enzymes in the snake venom is l-amino acid oxidase (LAAO) which plays an important role in the pathophysiological effects during snake envenomation. Some effects of this enzyme on the human body are apoptosis, platelet aggregation, edema, hemorrhage, and cytotoxicity. Hence, inhibiting the enzyme activity to reduce its degradation effects is of great medical and pharmacological importance. On the other hand, drug repurposing is a process to find the new existing drug for a new medical indication. Since Crotalus adamanteus LAAO has no crystal structure in the protein data bank, first, its 3D structure was constructed by homology modeling using 1REO as the template and then modeled structure was evaluated by several algorithms. We screened the FDA-approved drugs by structure-based virtual screening, molecular dynamics (MD) simulation, and Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) to identify new inhibitors for the snake venom LAAO. Interestingly, docking results revealed that half of the hits belong to the propionic acid derivatives drugs. In addition, MD simulation was performed to assess the interaction profile of the docked protein-hits complexes. Meanwhile, Arg88, Gln112, Lys345, Trp356 form consistent hydrogen bond interactions with Dexketoprofen, Flurbiprofen, Ketoprofen, Morphine, and Citric acid during simulation. According to the results, each of the four compounds can be an appropriate inhibitor of LAAO and since our study was based on drug repurposing could be evaluated in phase II clinical trials.

A comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms.

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action.

L-amino acid oxidase isolated from Micrurus mipartitus snake venom (MipLAAO) specifically induces apoptosis in acute lymphoblastic leukemia cells mostly via oxidative stress-dependent signaling mechanism.

The effect of Micrurus mipartitus snake venom as a therapeutic alternative for T-acute lymphoblastic leukemia (ALL) is still unknown. This study was aimed to evaluate the cytotoxic effect of M. mipartitus snake venom and a new L-amino acid oxidase (LAAO), named MipLAAO, on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat), and its mechanism of action. PBL and Jurkat cells were treated with venom and MipLAAO, and morphological changes in the cell nucleus/DNA, mitochondrial membrane potential, levels of intracellular reactive oxygen species and cellular apoptosis markers were determined by fluorescence microscopy, flow cytometry and pharmacological inhibition. Venom and MipLAAO induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner. Additionally, venom and MipLAAO increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, increased DJ-1 Cys106-sulfonate, as a marker of intracellular stress and induced the up-regulation of PUMA, p53 and phosphorylation of c-JUN. Additionally, it increased the expression of apoptotic CASPASE-3. In conclusion, M. mipartitus venom and MipLAAO selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway dependent mostly on CASPASE-3 pathway. Our findings support the potential use of M. mipartitus snake venom compounds as a potential treatment for T-ALL.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

The Pseudoalteromonas luteoviolacea L-amino Acid Oxidase with Antimicrobial Activity Is a Flavoenzyme.

The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress.

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

Role of l-amino acid oxidase isolated from Calloselasma rhodostoma venom on neutrophil NADPH oxidase complex activation.

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

Isolation and pharmacological characterization of a new cytotoxic L-amino acid oxidase from Bungarus multicinctus snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Bungarus multicinctus snake belongs to Elapidae family and is widely distributed in southern China. It is widely used in traditional Chinese medicine with the effect of dispelling wind and removing obstruction in the meridians. Moreover, it is also as a chief ingredient of many polyherbal formulations for the treatment of cancer. AIM OF THE STUDY: To evaluate the antitumor activity of Bungarus multicinctus snake venom components and isolate, characterize the most effective anti-tumor component of Bungarus multicinctus snake venom. MATERIALS AND METHODS: The in vitro antitumor activity of Bungarus multicinctus venom components was detected by cytotoxicity assay and cell apoptosis assay. A unique LAAO from Bungarus multicinctus venom named as BM-Apotxin was isolated and characterized by Sephadex G-75 gel filtration, Sephadex G-25 desalting, Q ion-exchange chromatography and subsequent amino acids sequence determination. The LAAO activity and enzyme kinetics of BM-Apotxin was detected by microplate assay. RESULTS: BM-Apotxin, a 65KDa glycoprotein, which contributed to the most anti-tumor effects of Bungarus multicinctus venom. BM-Apotxin can selectively kill tumor cells, with less cytotoxicity to the normal cells. BM-Apotxin is an L-amino acid oxidase (LAAO) with high sequence identity to other snake venom LAAOs. Its anti-tumor activity is mainly due to the hydrogen peroxide produced from LAAO oxidation. But the catalase did not reverse its anti-tumor effect completely. Like other snake venom LAAOs, BM-Apotxin can oxidize many L amino acids, not D amino acids. The optimum substrate for BM-Apotxin is L-Phe. Moreover, BM-Apotxin deglycosylation can significantly reduce the LAAO activity and anti-tumor activity of BM-Apotxin. CONCLUSION: This study will facilitate the study on anti-tumor mechanism of snake venom and drug development based on Bungarus multicinctus venom.

Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate.

l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom.

An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca2+, Mg2+ and Mn2+ did not alter Bpic-LAAO activity; however, Zn2+ is an inhibitor. Some reagents such as ß-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 µg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus.

A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities.

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56µg/mL) and Trypanosoma cruzi (IC50=4.85µg/mL). This toxin also induced cytotoxicity (IC50=1.80µg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Synergistic antibacterial effect of co-administering adipose-derived mesenchymal stromal cells and Ophiophagus hannah L-amino acid oxidase in a mouse model of methicillin-resistant Staphylococcus aureus-infected wounds.

BACKGROUND: Mesenchymal stromal cells (MSCs) and Ophiophagus hannah L-amino acid oxidase (Oh-LAAO) have been reported to exhibit antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). Published data have indicated that synergistic antibacterial effects could be achieved by co-administration of two or more antimicrobial agents. However, this hypothesis has not been proven in a cell- and protein-based combination. In this study, we investigate if co-administration of adipose-derived MSCs and Oh-LAAO into a mouse model of MRSA-infected wounds would be able to result in a synergistic antibacterial effect. METHODS: MSCs and Oh-LAAO were isolated and characterized by standard methodologies. The effects of the experimental therapies were evaluated in C57/BL6 mice. The animal study groups consisted of full-thickness uninfected and MRSA-infected wound models which received Oh-LAAO, MSCs, or both. Oh-LAAO was administered directly on the wound while MSCs were delivered via intradermal injections. The animals were housed individually with wound measurements taken on days 0, 3, and 7. Histological analyses and bacterial enumeration were performed on wound biopsies to determine the efficacy of each treatment. RESULTS: Immunophenotyping and differentiation assays conducted on isolated MSCs indicated expression of standard cell surface markers and plasticity which corresponds to published data. Characterization of Oh-LAAO by proteomics, enzymatic, and antibacterial assays confirmed the identity, purity, and functionality of the enzyme prior to use in our subsequent studies. Individual treatments with MSCs and Oh-LAAO in the infected model resulted in reduction of MRSA load by one order of magnitude to the approximate range of 6 log10 colony-forming units (CFU) compared to untreated controls (7.3 log10 CFU). Similar wound healing and improvements in histological parameters were observed between the two groups. Co-administration of MSCs and Oh-LAAO reduced bacterial burden by approximately two orders of magnitude to 5.1 log10 CFU. Wound closure measurements and histology analysis of biopsies obtained from the combinational therapy group indicated significant enhancement in the wound healing process compared to all other groups. CONCLUSIONS: We demonstrated that co-administration of MSCs and Oh-LAAO into a mouse model of MRSA-infected wounds exhibited a synergistic antibacterial effect which significantly reduced the bacterial count and accelerated the wound healing process.

Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.

L-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of L-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. L-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant L-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active L-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic L-amino acids as well as methyl esters of these L-amino acids. The progress of the conversion of L-phenylalanine and L-leucine into the corresponding α-keto acids was determined by HPLC and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50-100-fold by SDS treatment. K m values ranging from 0.9-10 mM and v max values from 3 to 10 U mg-1 were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant L-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.

Proteome and Peptidome of Vipera berus berus Venom.

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A2 and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A2 and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.

Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines.

An L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom was purified to homogeneity in a two-step procedure using molecular exclusion on Sephadex G-75, followed by Phenyl Sepharose FF chromatography. The molecular mass of the purified enzyme was 113 kDa, as determined by SDS-PAGE under reducing conditions. LAAOcdt showed amino acid homology to other L-amino acid oxidases isolated from different snake venoms. The comparative analysis of the internal peptide sequences of the NNPGILEYPVKPSEEGK fragments by LC-MS/MS spectrometry revealed 100% identity with C. durissus cumanensis LAAO. The purified protein catalyzed the oxidative deamination of L-amino acids, and the most specific substrates were L-Tyr and L-Phe. The enzyme presented optimum activity at pH 7.4 and at 44 °C. LAAOcdt also showed hemolytic activity (0.6-20 µg/µL) and induced both the formation plasma clots (5-100 µg/µL) and platelet aggregation (2.5 × 10(-3), 5.0 × 10(-3) and 10 × 10(-3) µg/mL), as well as bactericidal activity (2.5-10 µg/µL) against Staphylococcus aureus. Moreover, LAAOcdt exhibited cytotoxicity in distinct cancer cell lines, which presented a heterogeneous response profile. The mean IC50 value was 10.5 µg/mL. Glioma and pancreatic carcinoma cells were the most sensitive cell lines; they showed mean IC50 values of 7.2 µg/mL and 7.4 µg/mL, respectively. The exposure of the drug-sensitive cells to LAAOcdt for 24 h upregulated activated p-H2AX and efficiently decreased P42/P44 (ERK) activation in glioma cells (HCB151), which suggested an anti-proliferative effect. In addition, increased p21 expression was observed in SiHa cells, which showed a resistant phenotype. On the other hand, the flow cytometry and immunoblotting analyses showed that the enzyme did not induce cancer cell apoptosis. These results suggest that another cell death mechanism might contribute to the LAAOcdt-induced cytotoxicity. Taken together, this work may help to elucidate the function and structure of LAAOcdt by providing the basis for further investigations on its efficacy in cancer treatment.

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.

Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric l-amino acid oxidase (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics.