Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248 209

surface plasmon resonance to improve the molecularcharacterization of this heterocomplex.

Methods: Mass spectrometry (nanoESI-Q/TOF MS) wasused to analyze jararhagin, DM43 and the stoichiometry oftheir toxin-antitoxin complex; moreover, the quaternarystructure of DM43 was assessed by this methodology. Therate and equilibrium constants of the interaction weredetermined by surface plasmon resonance. The toxin wascaptured on a sensor chip derivatized with the anti-jar-arhaginmonoclonal antibodyMAJar 2 and the sensorgramsobtained after successive injections of DM43 in a concen-tration series were globally fitted to a simple bimolecularinteraction.

Results and Discussion: The stoichiometry of theinteraction was confirmed by MS; from native solutionconditions, the complex showed a molecular mass of w 94kDa, indicating that one molecule of jararhagin (50 kDa)interacts with one monomer of DM43 (43 kDa). Althoughreadily observed in solution, the dimeric structure of theinhibitor was barely preserved in the gas phase. This resultsuggests that, in contrast to the toxin-antitoxin complex,hydrophobic interactions are the primary driving force forthe inhibitor dimerization. Regarding the real-time inter-action analysis, the following kinetic rates, for the DM43/jararhagin interaction, were obtained: ka ¼ 3.54 � 0.03 �104 M-1s-1 and kd ¼ 1.16 � 0.07 � 10-5 s-1, resulting in anequilibrium dissociation constant (KD) of 0.33 � 0.06 nM.

Conclusions: The binding stoichiometry data deter-mined by MS unequivocally show that one monomer ofDM43 binds to one jararhaginmolecule. Moreover, we haveshown that DM43 dimers, which are rather stable in solu-tion, are not preserved in the gas phase corroborating thatits dimerization interfaces may be due to interactionbetween hydrophobic residues, a theoretical predictionbased on PATCHES analysis. Finally, the kinetic character-ization of this interaction indicates that DM43 binds to thereferred target toxin with high affinity and may constitutea suitable template for the rational development of novelhighly specific therapeutic agents to modulate the activityof SVMPs and their homologues.

Keywords: metalloproteinase, metalloproteinase inhibitor, snake venom,toxin, antitoxin, mass spectrometry, surface plasmon resonance10.1016/j.toxicon.2012.04.222

222. Molecular Mechanisms Involved in PGE2 ReleaseInduced by the Snake Venom Metalloproteinase BaP1 inSynoviocytes

Mariana Viana 1, Catarina Teixeira 1, Elbio Leiguez 1,José M. Gutiérrez 2, Alexandra Rucavado 2,Cristina M. Fernandes 11Unity of Inflammation, Laboratory of Pharmacology, Butantan Institute, SaoPaulo, Brazil2Clodomiro Picado Institute, University of Costa Rica, San José, CRE-mail address: catarinateixeira@butantan.gov.br (C.M. Fernandes).

Background: Snake venommetalloproteinases (SVMPs)and Matrix metalloproteinases (MMPs) share commondomain organization and exhibit identical Zn-bindingmotif. Studies on SVMPs may thus provide insights into the

functions of MMPs. Levels of these enzymes are increasedin inflamed articular joints. Articular synovial fibroblasts(B type) are the main cells involved in production andrelease of inflammatory mediators during joint inflamma-tory processes, being PGE2 a major mediator. BaP1 is a 22.7kDa SVMP from B. asper snake venom and contains only themetalloproteinase domain. This enzyme displays potentinflammatory activities both in vivo and in vitro experi-mental models. In this study we investigated the effects ofthis SVMP on isolated synoviocytes focusing on the releaseof prostaglandin E2 (PGE2) and the molecular mechanismsinvolved in this effect.

Methods: B type synoviocytes isolated from rat kneejoints synovial membranes (CEUIAB 576/09) were used.Levels of PGE2 were measured by enzyme immunoassayand protein expression of the PGE2 receptor EP4 wasdetermined by Western blotting. Participation of bothNF-kB and EP4 receptor in the release of PGE2 and COX-2protein expression induced by BaP1 was evaluated in cellspretreated with the inhibitors SN50 (50 mg/mL) andAH23848 (30 mM), respectively.

Results: BaP1 induced release of PGE2 from B typesynoviocytes after 1, 3, 6 and 12 h, but not 30 min incu-bation, in comparison with control cells incubated withRPMI alone. BaP1 induced EP4 protein expression (52 and65 kDa) by synoviocytes (30min – 6h). Moreover, inhibitionof NF-kB or EP4 receptor significantly decreased BaP1-induced PGE2 release and COX-2 protein expression.

Discussion: These data indicate the ability of BaP1 toinduce biosynthesis of PGE2 and COX-2 expression in iso-lated B type synoviocytes. These effects aremediated byNF-kB pathway. Moreover, data demonstrated that EP4receptor regulates BaP1-induced PGE2 production and COX-2 expression through a positive feedback loop, and stronglysuggest that this subtype of PGE2 receptor contributes foramplification of BaP1-induced release of PGE2.

Conclusion: BaP1 can directly stimulate synoviocytesfor synthesis of PGE2 and expression of both COX-2 enzymeand EP4 receptor. These findings suggest novelmechanismsof action displayed by metalloproteinases in synoviocytes.

Financial support: FAPESP; CNPq

Keywords: metalloproteinase, prostaglandin E2, synoviocyte10.1016/j.toxicon.2012.04.223

223. TLR2 and MyD88 Signaling are Required forEfficient Response of Macrophages to MT-IIIa Phospholipase A2 (PLA2) from Bothrops asper SnakeVenom

Elbio Leiguez 1, Karina C. Giannotti 1, Vanessa Moreira 1,Márcio H. Matsubara 1, Bruno Lomonte 2,Catarina F.P. Teixeira 1

1Butantan Institute, Laboratory of Pharmacology, Sao Paulo, Brazil2Clodomiro Picado Institute, University of Costa Rica, San José, CRE-mail address: catarinateixeira@butantan.gov.br (C.F.P. Teixeira).

Background: Toll-like receptors (TLRs) are majorcomponents of the innate immune system and primarysensors for noxious stimuli. Upon activation these