No abstract

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51–121.01 μM within single milked venom samples. This provides for a 6.37–20,965 fold-excess of toxin to induce apparent IC50 for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.

Currently, commercial plant peroxidases are all native and are isolated from plants such as horseradish and soybean. No recombinant plant peroxidase products have been available on the commercial market. The gene encoding peroxidase was cloned from windmill palm tree leaves. The codon-optimized gene was transformed into Pichia pastoris for expression. The recombinant windmill palm tree peroxidase (rWPTP) expressed by P. pastoris showed high stability under pH 2-10 and temperatures up to 70 °C to many metallic salts and organic solvents. The substrate specificity of WPTP was determined, and among the substrates tested, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was most suitable for WPTP. The Michaelis constants with the substrates HO and ABTS were 4.6 × 10 and 1.6 × 10 M, respectively. The rWPTP expressed in P. pastoris may be a suitable enzyme for the biosynthesis of polymers because of its high stability and activity under acidic conditions.

Plant secretory (Class III) peroxidases are redox enzymes that rely on N-glycosylation for full enzyme activity and stability. Peroxidases from palm tree leaves comprise the most stable and active plant peroxidases characterized to date. Herein, site-specific glycosylation and microheterogeneity of windmill palm tree (Trachycarpus fortunei) peroxidase are reported. The workflow developed in this study includes novel tools, written in R, to aid plant glycan identification, pGlycoFilter, for annotation of glycopeptide fragmentation spectra, gPSMvalidator, and for relative quantitation of glycoforms, glycoRQ. Mass spectrometry analysis provided a detailed glycosylation profile at the 13 sites of N-linked glycosylation on windmill palm tree peroxidase. Glycan microheterogeneity was observed at each site. Site Asn211 was the most heterogeneous and contained 30 different glycans. Relative quantitation revealed 90% of each glycosylation site was occupied by three or fewer glycans, and two of the 13 sites were partially unoccupied. Although complex and hybrid glycans were identified, the majority of glycans were paucimannosidic, characteristic of plant vacuolar glycoproteins. Further studies pertaining to the glycan structure-activity relationships in plant peroxidases can benefit from the work outlined here.

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai'i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD50 = 34.2 nMol Kg-1. The non-post-translationally modified form, [Pro]2,8[Glu]16α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4 ± 0.5 μM. Interestingly its comparative PD50 (3.6 μMol Kg-1) in invertebrates was ∼100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.