The small subunit of Hemilipin2, a new heterodimeric phospholipase A2 fromHemiscorpius lepturusscorpion venom, mediates the antiangiogenic effect of the whole protein


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Abstract

In a previous study, we reported the identification of Hemilipin, the first secreted heterodimeric phospholipase A2 (sPLA2) from Hemiscorpius lepturus scorpion venom and demonstrated its effective inhibition of all angiogenesis key steps in vitro and in vivo. Here, we aimed to characterize a second sPLA2, Hemilipin2, from the same venom and to elucidate its antiangiogenic effect. The protein was purified by chromatography separation and analyzed by MALDI/TOF mass spectrometry. Its N terminal amino acid sequence was determined by Edman degradation method and the enzymatic activity by fatty acids release assay. Hemilipin2 antiangiogenic activity was investigated by studying its effect in vitro on adhesion, migration and capillary like tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Pulmonary Artery Endothelial Cells (HPAECs); and in vivo on the chick embryo chorioallantoic membrane (CAM) assay. Data to be presented show that Hemilipin2 is heterodimeric composed by two subunits: the large one has a molecular weight of 12,866 and the small one of 2461 a.m.u. It has a strong calcium-dependent PLA2 activity and impacts angiogenesis in vitro and in vivo without showing any cytotoxic or apoptotic signs. Its chemical modification with p-Bromophenacyl Bromide abolishes the enzymatic activity without affecting the antiangiogenic effect. Furthermore, it has been proved that Hemilipin2 small subunit was able to inhibit blood vessel formation both in vitro and in vivo. These findings may serve as a starting point for the designing of a new generation of specific inhibitor of human angiogenesis at different steps.HIGHLIGHTSHemilipin2 is heterodimeric secreted phospholipase A2 (sPLA2) purified from Hemiscorpius lepturus scorpion.Hemilipin2's large and small subunits have a molecular mass of 12866 and 2461 a.m.u. respectively.Hemilipin2’s large subunit has no homology with other sPLA2s group III. Hemilipin2 could form a subgroup of group III sPLA2.Hemilipin2 exhibits an anti-angiogenesis activity on cells migration, adhesion and vessel length, and in vivo on CAM model.The antiangiogenic effect appears to be independent from the catalytic activity and seemingly held by the small subunit.

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