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LC–MS/MS Proteomic Profiling of Apis mellifera Venoms

ABSTRACT: This project presents a comparative proteomic analysis of crude bee venom collected from geographically and genetically distinct Apis mellifera populations from Türkiye and Cyprus. Bottom-up proteomics was performed using nanoLC–MS/MS on a high-resolution Orbitrap platform operating in data-dependent acquisition (DDA) mode. The study aimed to characterize regional differences in venom composition and to investigate potential genetic and post-translational variability in melittin, the dominant venom peptide. Proteomic profiling revealed that melittin is the predominant venom component across all populations, while regional discrimination is primarily driven by coordinated variation in moderately and low-abundance proteins, including phospholipase A2 (PLA2), apamin, secapin family peptides, and hyaluronidase. Targeted re-analysis of MS/MS spectra identified trace but reproducible O-linked glycosylation on melittin, representing the first evidence of naturally occurring O-glycosylation in this peptide. Label-free quantification was applied to assess relative protein abundances and glycoform distribution across regions.

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ORGANISM(S): Apis Mellifera (honeybee)

SUBMITTER: HACI MEHMET KAYILI

LAB HEAD: Hacı Mehmet

PROVIDER: PXD073864 | Pride | 2026-02-02

REPOSITORIES: Pride

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Project description:The Mojave rattlesnake (Crotalus scutulatus scutulatus) is classified as the “highest medically important” snake in the risk categories in the United States. Although responsible for fewer snakebite envenomations and deaths compared to other species, Mojave rattlesnake venom is poorly characterized and shows significant geographical variability. The venom of Type A animals primarily contains the β-neurotoxin referred to as Mojave Toxin (MTX), which is responsible for the neurotoxic effects that make bites from this snake particularly feared. Previous studies have shown that β-neurotoxin from different snake species produced similar but complex effects by mechanisms that are not fully understood. We performed a genome-wide transcriptomic analysis of the neurocellular response to Mojave Type A rattlesnake venom using induced pluripotent stem cell (iPSC) -derived human neural stem cells (NSCs) to unveil the molecular mechanisms underlying the damage caused by this snake’s envenomation. Our results suggest that snake venom metalloproteases (svMPs), although have a limited repertoire in type A animal venom, facilitate venom spread by digesting tissue's extracellular matrix. The MTX, which is composed of heterodimers of basic and acidic phospholipase A2 (PLA2) and is the dominant constituent of this venom, co-opts the host arachidonic acid and Ca2+ second messenger mechanisms in a dose- and time-dependent escalating venom damage. The release of arachidonic acid and the rapid increase in intracellular Ca2+ caused by the PLA2 activity of MTX triggers multiple signaling cascades. The activation of MAPKs and NF-κB regulated proinflammatory cascades were the top enriched pathways in the shorter 4-hour NSC response to venom challenge and suggest a significant role of PKC-δ in the activation of MAPKs. The rapid increase in intercellular Ca2+ and resulting cellular depolarization plausibly have a role in neurotransmitter overload in the cholinergic and glutamatergic excitatory synapses and MTX-induced presynaptic blockade of nerve signals. The expression of the acetylcholinesterase gene (ACHE), which degrades acetylcholine, and the downregulation of GRIK1 and GRIK3 genes, which encode KA-iGluRs proteins suggest a cellular response to neurotransmitter overload in the excitatory synapses. Our results also show that the MTX/svPLA2 mediated dysregulation of Ca2+ homeostasis, particularly depletion from the endoplasmic reticulum (ER), causes ER stress and upregulation of unfolded protein response (UPR). The UPR and the oxidative stress caused by ROS generated in CYP1A1-mediated hydroxylation of arachidonic acid, contribute to mitochondrial membrane permeabilization. The activation of UPR, mitochondrial toxicity, and oxidative stress, constitute the degenerative phase of the venom challenge in NSCs and synergistically contribute to apoptotic and ferroptotic programmed cell death.

Project description:Snake venom inhibitors have been studied for over a century. Among them are the plasma proteins of some mammals and of non-venomous and venomous snakes, which have the ability to promote resistance to the venom. These proteins have aroused biotechnological interest, as they are an excellent alternative for the development of new drugs. The main proteins found in animal plasma, capable of neutralizing venom, are metalloproteinase inhibitors and PLA2 inhibitors (PLIs). The PLA2 acts on cell membrane phospholipids resulting in fatty acids, lysophospholipids and cell membrane deconstruction. This protein is commonly responsible for the local effect of envenomation, causing tissue inflammation in the affected area. Based on this information, γBjPLI was recently isolated, which is a PLI present in the blood of the venomous snake Bothrops jararaca. This protein is classified as belonging to the gamma (γ) class, based on its characteristic domains. γBjPLI has approximately 22 kDa and inhibits the enzymatic, edematogenic and myonecrotic activities of PLA2, suggesting a role of this inhibitor in the protection of these snakes against self-envenomation. Knowing that, in this work it is presented a comparative study of the protein composition of the plasma of newborns, young and adults, females and males of B. jararaca, by analysis of SDS-PAGE, Western blotting, affinity chromatography, ELISA and mass spectrometry. Such analysis allowed to identify PLI-like proteins (αPLI, βPLI and γPLI) at all stages of life, however, there was no clear evidence that there is a significant difference in ontogeny. An evaluation of the interaction and neutralization of crotoxin anticoagulant activity by thromboelastometry was also performed, and γBjPLI was able to inhibit up to 43% of the activity. In addition, it showed a greater affinity for PLA2 Asp-49 compared to PLA2 Lys-49, evidenced by an alteration in spectra obtained by circular dichroism, and by the inhibition of myotoxic and edematogenic activities, with values of 19.6% and 38. 5% for PLA2 Asp-49, higher than 16% and 19.6% for PLA2 Lys-49, respectively. Furthermore, to establish new methods for obtaining these inhibitors with greater yield, stability, and activity, γBjPLI was cloned and expressed using the pET28a vector and a SHuffle expression strain of the bacterium species Escherichia coli

			Action	DRS
	AN_49_1.raw	Raw
	AN_49_2.raw	Raw
	AN_49_3.raw	Raw
	AN_A2_10_1.raw	Raw
	AN_A2_10_2.raw	Raw

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LC–MS/MS Proteomic Profiling of Apis mellifera Venoms

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