Fish-hunting cone snail disrupts prey's glucose homeostasis with weaponized mimetics of somatostatin and insulin.

Venomous animals have evolved diverse molecular mechanisms to incapacitate prey and defend against predators. Most venom components disrupt nervous, locomotor, and cardiovascular systems or cause tissue damage. The discovery that certain fish-hunting cone snails use weaponized insulins to induce hypoglycemic shock in prey highlights a unique example of toxins targeting glucose homeostasis. Here, we show that, in addition to insulins, the deadly fish hunter, Conus geographus, uses a selective somatostatin receptor 2 (SSTR2) agonist that blocks the release of the insulin-counteracting hormone glucagon, thereby exacerbating insulin-induced hypoglycemia in prey. The native toxin, Consomatin nG1, exists in several proteoforms with a minimized vertebrate somatostatin-like core motif connected to a heavily glycosylated N-terminal region. We demonstrate that the toxin's N-terminal tail closely mimics a glycosylated somatostatin from fish pancreas and is crucial for activating the fish SSTR2. Collectively, these findings provide a stunning example of chemical mimicry, highlight the combinatorial nature of venom components, and establish glucose homeostasis as an effective target for prey capture.

Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry.

Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.