Assessing marine ecosystem health using multi-omic analysis of blue mussel liquid biopsies: A case study within a national marine park.

Marine ecosystems are under escalating threats from myriad environmental stressors, necessitating a deeper understanding of their impact on biodiversity and the health of sentinel organisms. In this study, we carried out a spatiotemporal multi-omic analysis of liquid biopsies collected from mussels (Mytilus spp.) in marine ecosystems of a national park. We delved into the epigenomic, transcriptomic, glycomic, proteomic, and microbiomic profiles to unravel the intricate interplay between ecosystem biodiversity and mussels' biological response to their environments. Our analysis revealed temporal fluctuations in the alpha diversity of the circulating microbiome associated with human activities. Analysis of the hemolymphatic circulating cell-free DNA (ccfDNA) provided information on the biodiversity and the presence of potential pathogens. Epigenomic analysis revealed widespread hypomethylation sites within the mitochondrial (mtDNA). Comparative transcriptomic and glycomic analyses highlighted differences in metabolic pathways and genes associated with immune and wound healing functions. This study demonstrates the potential of multi-omic analysis of liquid biopsy in sentinel to provide a holistic view of human activities' environmental impacts on marine coastal ecosystems. Overall, this approach has the potential to enhance the effectiveness and efficiency of various conservation efforts, leading to more informed decision-making and better outcomes for biodiversity and ecosystem conservation.

Elusive Protein-Glycosphingolipid Interactions Revealed by Membrane Anchor-Assisted Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging. The present work introduces MEmbrane ANchor-assisted nMS (MEAN-nMS) for the detection of low-affinity GBP-GSL complexes. The assay utilizes a membrane anchor, produced by covalent cross-linking of the GBP and a lipid in the membrane, to localize the GBP on the surface and promote GSL binding. Ligands are identified by nMS detection of intact GBP-GSL complexes (MEAN-nMS) or using a catch-and-release (CaR) strategy, wherein GSLs are released from GBP-GSL complexes upon collisional activation and detected (MEAN-CaR-nMS). To establish reliability, a library of purified gangliosides incorporated into nanodiscs was screened against human immune lectins, and the results compared with affinities of the corresponding ganglioside oligosaccharides. Without a membrane anchor, nMS analysis yielded predominantly false negatives. In contrast, all ligands were identified by MEAN-(CaR)-nMS, with no false positives. To highlight the potential of MEAN-CaR-nMS for ligand discovery, a natural library of GSLs was incorporated into nanodiscs and screened against human and viral proteins to uncover elusive ligands. Finally, nMS-based detection of GSL ligands directly from cells is demonstrated. This breakthrough paves the way for shotgun glycomics screening using intact cells.

Detection of Toxoplasma gondii in wild bivalves from the Kerguelen and Galapagos archipelagos: influence of proximity to cat populations, exposure to marine currents and kelp density.

Oocysts of the protozoan Toxoplasma gondii are found in felid feces and can be washed into coastal waters, where they persist for months, attaching to algae and accumulating in invertebrates. We used wild bivalves to assess contamination of coastal waters of the Kerguelen and Galapagos archipelagos by this zoonotic parasite. Additionally, we leveraged the contrasting situations of these archipelagos to identify some potential drivers of contamination. In the Galapagos, with a cat density reaching 142 per km2, 15.38% of the sampled oysters (Saccostrea palmula) tested positive for T. gondii by quantitative real-time PCR (qPCR) (n = 260), and positive samples were found in all eight sampling sites. In Kerguelen, with 1-3 cats per km2, 40.83% of 120 tested mussels (Mytilus edulis platensis) were positive, and positive samples were found in four out of the five sampling sites. These findings provide evidence of T. gondii contamination in the coastal waters of these archipelagos. Furthermore, T. gondii-positive bivalves were found on islands located 20 km away (Galapagos) and 5 km away (Kerguelen) from the nearest cat population, indicating that T. gondii oocysts can disperse through waterborne mechanisms over several kilometers from their initial deposition site. In the Galapagos, where runoff is infrequent and all sites are exposed to currents, the prevalence of qPCR-positive bivalves did not show significant variations between sites (p = 0.107). In Kerguelen where runoff is frequent and site exposure variable, the prevalence varied significantly (p < 0.001). The detection of T. gondii in Kerguelen mussels was significantly correlated with the site exposure to currents (odds ratio (OR) 60.2, p < 0.001) and the on-site density of giant kelp forests (OR 2.624, p < 0.001). This suggests that bivalves can be contaminated not only by oocysts transported by currents but also by consuming marine aggregates containing oocysts that tend to form in kelp forests.

From shells to sequences: A proof-of-concept study for on-site analysis of hemolymphatic circulating cell-free DNA from sentinel mussels using Nanopore technology.

Blue mussels are often abundant and widely distributed in polar marine coastal ecosystems. Because of their wide distribution, ecological importance, and relatively stationary lifestyle, bivalves have long been considered suitable indicators of ecosystem health and changes. Monitoring the population dynamics of blue mussels can provide information on the overall biodiversity, species interactions, and ecosystem functioning. In the present work, we combined the concept of liquid biopsy (LB), an emerging concept in medicine based on the sequencing of free circulating DNA, with the Oxford Nanopore Technologies (ONT) platform using a portable laboratory in a remote area. Our results demonstrate that this platform is ideally suited for sequencing hemolymphatic circulating cell-free DNA (ccfDNA) fragments found in blue mussels. The percentage of non-self ccfDNA accounted for >50 % of ccfDNA at certain sampling Sites, allowing the quick, on-site acquisition of a global view of the biodiversity of a coastal marine ecosystem. These ccfDNA fragments originated from viruses, bacteria, plants, arthropods, algae, and multiple Chordata. Aside from non-self ccfDNA, we found DNA fragments from all 14 blue mussel chromosomes, as well as those originating from the mitochondrial genomes. However, the distribution of nuclear and mitochondrial DNA was significantly different between Sites. Similarly, analyses between various sampling Sites showed that the biodiversity varied significantly within microhabitats. Our work shows that the ONT platform is well-suited for LB in sentinel blue mussels in remote and challenging conditions, enabling faster fieldwork for conservation strategies and resource management in diverse settings.

Divergent bacterial landscapes: unraveling geographically driven microbiomes in Atlantic cod.

Establishing microbiome signatures is now recognized as a critical step toward identifying genetic and environmental factors shaping animal-associated microbiomes and informing the health status of a given host. In the present work, we prospectively collected 63 blood samples of the Atlantic cod population of the Southern Gulf of Saint Lawrence (GSL) and characterized their 16S rRNA circulating microbiome signature. Our results revealed that the blood microbiome signature was dominated at the phylum level by Proteobacteria, Bacteroidetes, Acidobacteria and Actinobacteria, a typical signature for fish populations inhabiting the GSL and other marine ecosystems. At the genus level, however, we identified two distinct cod groups. While the microbiome signature of the first group was dominated by Pseudoalteromonas, a genus we previously found in the microbiome signature of Greenland and Atlantic halibut populations of the GSL, the second group had a microbiome signature dominated by Nitrobacter and Sediminibacterium (approximately 75% of the circulating microbiome). Cods harboring a Nitrobacter/Sediminibacterium-rich microbiome signature were localized in the most southern part of the GSL, just along the northern coast of Cape Breton Island. Atlantic cod microbiome signatures did not correlate with the weight, length, relative condition, depth, temperature, sex, and salinity, as previously observed in the halibut populations. Our study provides, for the first time, a unique snapshot of the circulating microbiome signature of Atlantic cod populations and the potential existence of dysbiotic signatures associated with the geographical distribution of the population, probably linked with the presence of nitrite in the environment.

Targeting intracellular galectins for cancer treatment.

Although considerable attention has been paid to the role of extracellular galectins in modulating, positively or negatively, tumor growth and metastasis, we have witnessed a growing interest in the role of intracellular galectins in response to their environment. This is not surprising as many galectins preferentially exist in cytosolic and nuclear compartments, which is consistent with the fact that they are exported outside the cells via a yet undefined non-classical mechanism. This review summarizes our most recent knowledge of their intracellular functions in cancer cells and provides some directions for future strategies to inhibit their role in cancer progression.

Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry.

Native mass spectrometry (nMS) screening of natural glycan libraries against glycan-binding proteins (GBPs) is a powerful tool for ligand discovery. However, as the glycan concentrations are unknown, affinities cannot be measured directly from natural libraries. Here, we introduce Concentration-Independent (COIN)-nMS, which enables quantitative screening of natural glycan libraries by exploiting slow mixing of solutions inside a nanoflow electrospray ionization emitter. The affinities (Kd) of detected GBP-glycan interactions are determined, simultaneously, from nMS analysis of their time-dependent relative abundance changes. We establish the reliability of COIN-nMS using interactions between purified glycans and GBPs with known Kd values. We also demonstrate the implementation of COIN-nMS using the catch-and-release (CaR)-nMS assay for glycosylated GBPs. The COIN-CaR-nMS results obtained for plant, fungal, viral, and human lectins with natural libraries containing hundreds of N-glycans and glycopeptides highlight the assay's versatility for discovering new ligands, precisely measuring their affinities, and uncovering "fine" specificities. Notably, the COIN-CaR-nMS results clarify the sialoglycan binding properties of the SARS-CoV-2 receptor binding domain and establish the recognition of monosialylated hybrid and biantennary N-glycans. Moreover, pharmacological depletion of host complex N-glycans reduces both pseudotyped virions and SARS-CoV-2 cell entry, suggesting that complex N-glycans may serve as attachment factors.

Insights into the circulating microbiome of Atlantic and Greenland halibut populations: the role of species-specific and environmental factors.

Establishing long-term microbiome-based monitoring programs is critical for managing and conserving wild fish populations in response to climate change. In most cases, these studies have been conducted on gut and, to a lesser extent, skin (mucus) microbiomes. Here, we exploited the concept of liquid biopsy to study the circulating bacterial microbiome of two Northern halibut species of economic and ecological importance. Amplification and sequencing of the 16S rRNA gene were achieved using a single drop of blood fixed on FTA cards to identify the core blood microbiome of Atlantic and Greenland halibut populations inhabiting the Gulf of St. Lawrence, Canada. We provide evidence that the circulating microbiome DNA (cmDNA) is driven by genetic and environmental factors. More specifically, we found that the circulating microbiome signatures are species-specific and vary according to sex, size, temperature, condition factor, and geographical localization. Overall, our study provides a novel approach for detecting dysbiosis signatures and the risk of disease in wild fish populations for fisheries management, most notably in the context of climate change.

Placental Galectins in Cancer: Why We Should Pay More Attention.

The first studies suggesting that abnormal expression of galectins is associated with cancer were published more than 30 years ago. Today, the role of galectins in cancer is relatively well established. We know that galectins play an active role in many types of cancer by regulating cell growth, conferring cell death resistance, or inducing local and systemic immunosuppression, allowing tumor cells to escape the host immune response. However, most of these studies have focused on very few galectins, most notably galectin-1 and galectin-3, and more recently, galectin-7 and galectin-9. Whether other galectins play a role in cancer remains unclear. This is particularly true for placental galectins, a subgroup that includes galectin-13, -14, and -16. The role of these galectins in placental development has been well described, and excellent reviews on their role during pregnancy have been published. At first sight, it was considered unlikely that placental galectins were involved in cancer. Yet, placentation and cancer progression share several cellular and molecular features, including cell invasion, immune tolerance and vascular remodeling. The development of new research tools and the concomitant increase in database repositories for high throughput gene expression data of normal and cancer tissues provide a new opportunity to examine the potential involvement of placental galectins in cancer. In this review, we discuss the possible roles of placental galectins in cancer progression and why they should be considered in cancer studies. We also address challenges associated with developing novel research tools to investigate their protumorigenic functions and design highly specific therapeutic drugs.

Galectin-7 dysregulates renin-angiotensin-aldosterone and NADPH oxide synthase pathways in preeclampsia.

OBJECTIVES: Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Poor placentation in the first trimester of pregnancy is widely accepted to be an underlying cause of preeclampsia. Galectin-7 is abnormally elevated in chorionic villous samples and serum from women that subsequently develop pre-term preeclampsia. Administration of exogenous galectin-7 to pregnant mice causes preeclampsia-like features (hypertension, proteinuria), associated with dysregulation of the renin-angiotensin system (RAS). In this study investigated the mechanism by which galectin-7 induces alterations to tissue RAS homeostasis and ROS production. We hypothesized that galectin-7 induces alterations in the production of either placental RAS or NADPH oxidases (or both) to drive the dysregulated RAS and ROS production seen in preeclampsia. STUDY DESIGN: Mated female mice (n = 5-6/group) received single (embryonic day [E]12/13) or multiple (E8-12) subcutaneous injections of 400 µg/kg/day galectin-7 or vehicle control and killed on E13 or E18. Human first trimester placental villous and decidual tissue (n = 11) was cultured under 8 % oxygen with 1 µg/mL galectin-7 or vehicle control for 16 h. RESULTS: Galectin-7 administration to pregnant mice impaired placental labyrinth formation, suppressed circulating aldosterone and altered placental RAS (Agt, Renin) and NADPH oxidase (Cyba, Cybb and Icam1) mRNA expression. In vitro, galectin-7 regulated human placental villous RAS (AGT) and NADPH oxidase (CYBA, ICAM1 and VCAM1) mRNA expression. CONCLUSIONS: Overall, galectin-7 likely drives hypertension in preeclampsia via its direct regulation of multiple pathways associated with preeclampsia in the placenta. Galectin-7 may therefore be a therapeutic target to improve placental function and prevent preeclampsia.

Quantifying Biomolecular Interactions Using Slow Mixing Mode (SLOMO) Nanoflow ESI-MS.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes in vitro and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs). For many biomolecular complexes, such as protein-protein interactions, this condition is not satisfied. Existing strategies to correct for nonuniform RFs are generally incompatible with static nanoflow ESI (nanoESI) sources, which are typically used for biomolecular interaction studies, thereby significantly limiting the utility of ESI-MS. Here, we introduce slow mixing mode (SLOMO) nanoESI-MS, a direct technique that allows both the RF and affinity (K d) for a biomolecular interaction to be determined from a single measurement using static nanoESI. The approach relies on the continuous monitoring of interacting species and their complexes under nonhomogeneous solution conditions. Changes in ion signals of free and bound species as the system approaches or moves away from a steady-state condition allow the relative RFs of the free and bound species to be determined. Combining the relative RF and the relative abundances measured under equilibrium conditions enables the K d to be calculated. The reliability of SLOMO and its ease of use is demonstrated through affinity measurements performed on peptide-antibiotic, protease-protein inhibitor, and protein oligomerization systems. Finally, affinities measured for the binding of human and bacterial lectins to a nanobody, a viral glycoprotein, and glycolipids displayed within a model membrane highlight the tremendous power and versatility of SLOMO for accurately quantifying a wide range of biomolecular interactions important to human health and disease.

Species- and site-specific circulating bacterial DNA in Subantarctic sentinel mussels Aulacomya atra and Mytilus platensis.

Impacts of climate changes are particularly severe in polar regions where warmer temperatures and reductions in sea-ice covers threaten the ecological integrity of marine coastal ecosystems. Because of their wide distribution and their ecological importance, mussels are currently used as sentinel organisms in monitoring programs of coastal ecosystems around the world. In the present study, we exploited the concept of liquid biopsy combined to a logistically friendly sampling method to study the hemolymphatic bacterial microbiome in two mussel species (Aulacomya atra and Mytilus platensis) in Kerguelen Islands, a remote Subantarctic volcanic archipelago. We found that the circulating microbiome signatures of both species differ significantly even though their share the same mussel beds. We also found that the microbiome differs significantly between sampling sites, often correlating with the particularity of the ecosystem. Predictive models also revealed that both species have distinct functional microbiota, and that the circulating microbiome of Aulacomya atra was more sensitive to changes induced by acute thermal stress when compared to Mytilus platensis. Taken together, our study suggests that defining circulating microbiome is a useful tool to assess the health status of marine ecosystems and to better understand the interactions between the sentinel species and their habitat.

Applying the concept of liquid biopsy to monitor the microbial biodiversity of marine coastal ecosystems.

Liquid biopsy (LB) is a concept that is rapidly gaining ground in the biomedical field. Its concept is largely based on the detection of circulating cell-free DNA (ccfDNA) fragments that are mostly released as small fragments following cell death in various tissues. A small percentage of these fragments are from foreign (nonself) tissues or organisms. In the present work, we applied this concept to mussels, a sentinel species known for its high filtration capacity of seawater. We exploited the capacity of mussels to be used as natural filters to capture environmental DNA fragments of different origins to provide information on the biodiversity of marine coastal ecosystems. Our results showed that hemolymph of mussels contains DNA fragments that varied considerably in size, ranging from 1 to 5 kb. Shotgun sequencing revealed that a significant amount of DNA fragments had a nonself microbial origin. Among these, we found DNA fragments derived from bacteria, archaea, and viruses, including viruses known to infect a variety of hosts that commonly populate coastal marine ecosystems. Taken together, our study shows that the concept of LB applied to mussels provides a rich and yet unexplored source of knowledge regarding the microbial biodiversity of a marine coastal ecosystem.

Galectins in Glioma: Current Roles in Cancer Progression and Future Directions for Improving Treatment.

Traditional wisdom suggests that galectins play pivotal roles at different steps in cancer progression. Galectins are particularly well known for their ability to increase the invasiveness of cancer cells and their resistance to drug-induced cell death. They also contribute to the development of local and systemic immunosuppression, allowing cancer cells to escape the host's immunological defense. This is particularly true in glioma, the most common primary intracranial tumor. Abnormally high production of extracellular galectins in glioma contributes to the establishment of a strong immunosuppressive environment that favors immune escape and tumor progression. Considering the recent development and success of immunotherapy in halting cancer progression, it is logical to foresee that galectin-specific drugs may help to improve the success rate of immunotherapy for glioma. This provides a new perspective to target galectins, whose intracellular roles in cancer progression have already been investigated thoroughly. In this review, we discuss the mechanisms of action of galectins at different steps of glioma progression and the potential of galectin-specific drugs for the treatment of glioma.

Perturbing dimer interactions and allosteric communication modulates the immunosuppressive activity of human galectin-7.

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.

Towards a Better Understanding of the Relationships between Galectin-7, p53 and MMP-9 during Cancer Progression.

It has been almost 25 years since the discovery of galectin-7. This member of the galectin family has attracted interest from many working in the cancer field given its highly restricted expression profile in epithelial cells and the fact that cancers of epithelial origin (carcinoma) are among the most frequent and deadly cancer subtypes. Initially described as a p53-induced gene and associated with apoptosis, galectin-7 is now recognized as having a protumorigenic role in many cancer types. Several studies have indeed shown that galectin-7 is associated with aggressive behavior of cancer cells and induces expression of MMP-9, a member of the matrix metalloproteinases (MMP) family known to confer invasive behavior to cancer cells. It is therefore not surprising that many studies have examined its relationships with p53 and MMP-9. However, the relationships between galectin-7 and p53 and MMP-9 are not always clear. This is largely because p53 is often mutated in cancer cells and such mutations drastically change its functions and, consequently, its association with galectin-7. In this review, we discuss the functional relationships between galectin-7, p53 and MMP-9 and reconcile some apparently contradictory observations. A better understanding of these relationships will help to develop a working hypothesis and model that will provide the basis for further research in the hope of establishing a new paradigm for tackling the role of galectin-7 in cancer.

Hemocytes released in seawater act as Trojan horses for spreading of bacterial infections in mussels.

Global warming has been associated with increased episodes of mass mortality events in invertebrates, most notably in bivalves. Although the spread of pathogens is one of multiple factors that contribute to such mass mortality events, we don't fully understand the pathophysiological consequences of sea warming on invertebrates. In this work, we show that in temperature stress conditions, circulating hemocytes in mussels leave the hemolymph to gain access to the intervalvar fluid before being released in seawater. External hemocytes can survive for several hours in seawater before entering other mussels. When infected by bacteria, externally-infected hemocytes can enter naive mussels and promote bacterial dissemination in the host. These results reveal the existence of a new opportunistic mechanism used by pathogens to disseminate in marine ecosystems. Such mechanisms may explain how thermal anomalies triggered by global warming can favor episodic mass mortality observed in recent years in marine ecosystem.

Binding of a Soluble meso-Tetraarylporphyrin to Human Galectin-7 Induces Oligomerization and Modulates Its Pro-Apoptotic Activity.

The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.

Galectin-7 Impairs Placentation and Causes Preeclampsia Features in Mice.

Preeclampsia is a serious pregnancy-induced disorder unique to humans. The etiology of preeclampsia is poorly understood; however, poor placental formation is thought causal. Galectin-7 is produced by trophoblast and is elevated in first-trimester serum of women who subsequently develop preeclampsia. We hypothesized that elevated placental galectin-7 may be causative of preeclampsia. Here, we demonstrated increased galectin-7 production in chorionic villous samples from women who subsequently develop preterm preeclampsia compared with uncomplicated pregnancies. In vitro, galectin-7 impaired human first-trimester trophoblast outgrowth, increased placental production of the antiangiogenic sFlt-1 splice variant, sFlt-1-e15a, and reduced placental production and secretion of ADAM12 (a disintegrin and metalloproteinase12) and angiotensinogen. In vivo, galectin-7 administration (E8-E12) to pregnant mice caused elevated systolic blood pressure, albuminuria, impaired placentation (reduced labyrinth vascular branching, impaired decidual spiral artery remodeling, and a proinflammatory placental state demonstrated by elevated IL1ß, IL6 and reduced IL10), and dysregulated expression of renin-angiotensin system components in the placenta, decidua, and kidney, including angiotensinogen, prorenin, and the angiotensin II type 1 receptor. Collectively, this study demonstrates that elevated galectin-7 during placental formation contributes to abnormal placentation and suggests that it leads to the development of preeclampsia via altering placental production of sFlt-1 and renin-angiotensin system components. Targeting galectin-7 may be a new treatment option for preeclampsia.

Neoglycolipids as Glycosphingolipid Surrogates for Protein Binding Studies Using Nanodiscs and Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) in the membranes of cells are implicated in a wide variety of normal and pathophysiological processes. Despite the critical biological roles these interactions play, the GSL ligands of most GBPs have not yet been identified. The limited availability of purified GSLs represents a significant challenge to the discovery and characterization of biologically relevant GBP-GSL interactions. The present work investigates the use of neoglycolipids (NGLs) as surrogates for GSLs for catch-and-release-electrospray ionization mass spectrometry (CaR-ESI-MS)-based screening, implemented with nanodiscs, for the discovery of GSL ligands. Three pairs of NGLs based on the blood group type A and B trisaccharides, with three different lipid head groups but all with "ring-closed" monosaccharide residue at the reducing end, were synthesized. The incorporation efficiencies (into nanodiscs) of the NGLs and their affinities for a fragment of family 51 carbohydrate-binding module (CBM) identified an amide-linked 1,3-di-O-hexadecyl-glycerol moiety as the optimal lipid structure. Binding measurements performed on cholera toxin B subunit homopentamer (CTB5) and nanodiscs containing an NGL consisting of the optimal lipid moiety and the GM1 ganglioside pentasaccharide yielded affinities similar, within a factor of 2, to those of native GM1. Finally, nanodiscs containing the optimal A and B trisaccharide NGLs, as well as the corresponding NGLs of lactose, A type 2 tetrasaccharide, and the GM1 and GD2 pentasaccharides were screened against the family 51 CBM, human galectin-7, and CTB5 to illustrate the potential of NGLs to accelerate the discovery of GSL ligands of GBPs.