Functional Characterization of the Nemertide α Family of Peptide Toxins

Abstract

Peptide toxins find use in medicine, biotechnology, and agriculture. They are exploited as pharmaceutical tools, particularly for the investigation of ion channels. Here, we report the synthesis and activity of a novel family of peptide toxins: the cystine-knotted α nemertides. Following the prototypic α-1 and -2 ( 1 and 2 ), six more nemertides were discovered by mining of available nemertean transcriptomes. Here, we describe their synthesis using solid phase peptide chemistry and their oxidative folding by using an improved protocol. Nemertides α-2 to α-7 ( 2 – 7 ) were produced to characterize their effect on voltage-gated sodium channels ( Blatella germanica BgNa V 1 and mammalian Na V s1.1–1.8). In addition, ion channel activities were matched to in vivo tests using an Artemia microwell assay. Although nemertides demonstrate high sequence similarity, they display variability in activity on the tested Na V s. The nemertides are all highly toxic to Artemia , with EC 50 values in the sub-low micromolar range, and all manifest preference for the insect BgNa V 1 channel. Structure–activity relationship analysis revealed key residues for Na V -subtype selectivity. Combined with low EC 50 values (e.g., Na V 1.1: 7.9 nM (α-6); Na V 1.3: 9.4 nM (α-5); Na V 1.4: 14.6 nM (α-4)) this underscores the potential utility of α-nemertides for rational optimization to improve selectivity.

Microbial Transformation of Hesperidin and Biological Evaluation

“…The Artemia microwell assay was modified according to the Jacobsson method. 52 Artemia cysts (Artemio pur, JBL, Neuhofen, Germany) were hatched in a separation funnel containing artificial seawater (33 g of salt/L, Coral pro salt, Red Sea, Eilat, Israel) prepared in deionized water. A small aquarium air pump was used to aerate the water until the shrimp were harvested (24 h, RT).…”

Venomous Noodles: evolution of toxins in Nemertea through positive selection and gene duplication

“…We selected the gene families most likely to be toxins for further analyses. The cytotoxin-A and the alpha-nemertides had their toxicity well described in previous studies (Kem and Blumenthal 1978;Berne et al 2003;Jacobsson et al 2018;Jacobsson et al 2021). Although no biochemical description of the beta-nemertides has been made to our knowledge, its presence in the mucus with similarity to the Neurotoxin-BII, which has been biochemically characterized (Kem 1976), made Jacobsson et al (2018) consider it a toxin.…”

“…A thoroughly functional characterization of these toxins' family has described the effects of different alphanemertides in Na-channels. However, the role of these peptides in envenomation has yet to be fully elucidated (Jacobsson et al 2021). Looking closer at the alignment, we can also observe that sequences belonging to different clades differ in the C-Terminal segment, indicating that the product protein may have different properties (Figure S8).…”

Peptide Toxins from Antarctica: The Nemertean Predator and Scavenger Parborlasia corrugatus (McIntosh, 1876)

“…Interestingly, the 3718 Da peptide (c3718 in Figure 4) corresponds to the contig 3716Da_744_c37678 sequence by mass (average 1 z vs. predicted monoisotopic) and number of Cys residues. Moreover, the shift in retention to a more hydrophilic region in the chromatogram after alkylation is indicative of a native constrained structure with hydrophobic residues forced to the surface, i.e., as with alpha nemertides [31] and the 3624 Da peptide, indicating similar structural features.…”