Biology subjects

Salvage, S. C.

Publications and source records attributed to Salvage, S. C..

5 recordsLinked to original sources

bioRxiv2025

A robust platform for recombinant production of animal venom toxin modulators of ion channels

Background and PurposePeptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions. Experimental Approach13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca2+ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding. Key ResultsMonovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK) toxins from spiders, and -toxins from scorpions, substantially surpassing the performance of unfused toxins or bivalent Fc-toxin fusions which gave low or no yield. Snake toxins targeting nicotinic acetylcholine receptors retained high single digit nanomolar inhibitory potency. Spider and scorpion toxins targeting the voltage-gated Na+ channel Nav1.7 retained pharmacological function and selectivity across a panel of five Nav subtypes, albeit with reduced potencies that did not exceed [~]70 nM. Conclusions and ImplicationsWe present a strategy for straightforward robust production of pure, monodisperse, and functional animal venom-derived toxins. This lowers the barrier to toxin production in a standard laboratory setting for follow-on engineering purposes.

pharmacology and toxicology↗

bioRxiv2025

Structural implications of glycosylation on the voltage-gated sodium channel β3-subunit

Voltage-gated sodium (NaV) channel -subunits are modulated by associated {beta}-subunits that affect their localization, trafficking and gating behaviour. The {beta}-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length {beta}3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric {beta}3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated {beta}3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of {beta}3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the {beta}3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length {beta}3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length {beta}3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric {beta}-subunits with corresponding -subunit sodium channels.

biochemistry↗

bioRxiv2024

Isoform-specific N-linked glycosylation of voltage-gated sodium channel alpha-subunits alters beta-subunit binding sites

HighlightsO_LIThree N-linked glycosylation sites conserved among all Nav channels C_LIO_LIGlycan modelling and molecular dynamics simulations highlight 3D landscape C_LIO_LIUnique Nav1.5 N-linked glycans may prevent binding to Ig-domains of {beta}1 and {beta}3 C_LIO_LIUnique Nav1.8 N-linked glycan may prevent binding to Ig-domains of {beta}2 and {beta}4 C_LIO_LIN-linked glycans likely contribute to supra-molecular clustering of Nav channels C_LI Voltage-gated sodium channel -subunits (Nav1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The Nav {beta}-subunits ({beta}1-4) have been shown to modulate -subunit properties. Homo-oligomerization of {beta}-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several Nav channel isoforms and {beta}-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the Nav cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the Nav channels. Herein, molecular modelling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on Nav channel surfaces. The simulations revealed that negatively-charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two Nav1.5 isoform-specific glycans extensively cover the -subunit region that, in other Nav channel -subunit isoforms, corresponds to the binding site for the {beta}1-(and likely {beta}3-) subunit immunoglobulin (Ig) domain. Nav1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the {beta}2 and {beta}4-subunit Ig domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example by redirecting {beta}-subunit Ig-domains outwards to permit cis or trans supra-clustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.

biochemistry↗

bioRxiv2023

An Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knock down of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases glycolysis in breast cancer cells, likely by up-regulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

cancer biology↗

bioRxiv2021

Pathological turret mutations in the cardiac sodium channel cause long-range pore disruption

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Germline mutations that disrupt Nav1.5 activity predispose affected individuals to inherited cardiopathologies. Some of these Nav1.5 mutations alter amino acids in extracellular turret domains DII and DIII. Yet the mechanism is unclear. In the rat Nav1.5 structure determined by cryogenic electron microscopy, the wild-type residues corresponding to these mutants form a complex salt-bridge between the DII and DIII turret interface. Furthermore, adjacent aromatic residues form cation-{pi} interactions with the complex salt-bridge. Here, we examine this region using site-directed mutagenesis, electrophysiology and in silico modeling. We confirm functional roles for the salt-bridges and the aromatic residues. We show that their disruption perturbs the geometry of both the DEKA selectivity ring and the inner pore vestibule that are crucial for sodium ion permeability. Our findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms too. Our work illustrates how the sodium channel structures now being reported can be used to formulate and guide novel functional hypotheses.

biochemistry↗