ABSTRACT
More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.
Subject(s)
Cell Culture Techniques/methods , Organoids/growth & development , Snake Venoms/biosynthesis , Animals , Antivenins/genetics , In Situ Hybridization, Fluorescence/methods , Snake Venoms/chemistry , Snake Venoms/genetics , Snakes/geneticsABSTRACT
The Harderian gland is a cephalic structure, widely distributed among vertebrates. In snakes, the Harderian gland is anatomically connected to the vomeronasal organ via the nasolacrimal duct, and in some species can be larger than the eyes. The function of the Harderian gland remains elusive, but it has been proposed to play a role in the production of saliva, pheromones, thermoregulatory lipids and growth factors, among others. Here, we have profiled the transcriptomes of the Harderian glands of three non-front-fanged colubroid snakes from Cuba: Caraiba andreae (Cuban Lesser Racer); Cubophis cantherigerus (Cuban Racer); and Tretanorhinus variabilis (Caribbean Water Snake), using Illumina HiSeq2000 100â¯bp paired-end. In addition to ribosomal and non-characterized proteins, the most abundant transcripts encode putative transport/binding, lipocalin/lipocalin-like, and bactericidal/permeability-increasing-like proteins. Transcripts coding for putative canonical toxins described in venomous snakes were also identified. This transcriptional profile suggests a more complex function than previously recognized for this enigmatic organ.
Subject(s)
Colubridae/metabolism , Gene Expression Regulation/physiology , Harderian Gland/metabolism , Reptilian Proteins/biosynthesis , Snake Venoms/biosynthesis , Transcriptome/physiology , Animals , Colubridae/genetics , Cuba , Reptilian Proteins/genetics , Snake Venoms/geneticsABSTRACT
Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3-7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation , RNA Stability/genetics , Snake Venoms/biosynthesis , Snake Venoms/genetics , Animal Structures , Animals , Chromatography, High Pressure Liquid , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Freeze Drying , Mass Spectrometry , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Snake Venoms/chemistry , Snake Venoms/metabolismABSTRACT
The alpha(5)beta(1) integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the alpha(5)beta(1) integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC(50) of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC(50) of 3.75 microM. K562 cells bind to BaG-I probably through interaction with alpha(5)beta(1) integrin, since anti-alpha(5)beta(1) antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the alpha(5)beta(1) integrin.
Subject(s)
Disintegrins/chemistry , Disintegrins/isolation & purification , Metalloendopeptidases/chemistry , Metalloendopeptidases/isolation & purification , Amino Acid Sequence , Animals , Bothrops/classification , Bothrops/metabolism , Cell Adhesion/drug effects , Dimerization , Disintegrins/biosynthesis , Disintegrins/pharmacology , Fibronectins/metabolism , Humans , Integrin alpha5beta1/metabolism , K562 Cells/drug effects , K562 Cells/physiology , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/pharmacology , Molecular Sequence Data , Molecular Weight , Platelet Aggregation/drug effects , Protein Binding/drug effects , Snake Venoms/biosynthesis , Snake Venoms/chemistry , Snake Venoms/isolation & purification , Snake Venoms/pharmacology , Species SpecificityABSTRACT
A subfamília reprolisina de metaloproteinases inclui metaloproteinases veneno de serpente (SMVP) e desintegrina mamíferos / metaloproteinase. Estas proteínas são sintetizadas como zimogénios e submetidos a processamento proteolítico, resultando em uma variedade de proteínas multifuncionais.