Lectin Binding Research Articles

High-throughput sequencing reveals the existence of a novel fucose binding lectin gene from the whole gut and fat body tissues of the grub of darkling beetle, Zophobas morio

Fucose binding lectins are a taxonomically, evolutionarily, and ecologically relevant class of lectins that have been identified among a wide range of taxa. These lectins possess a distinctive ability to identify and bind fucose-containing glycans, thereby lending them the ability to function as immune cell mitogens, diagnostic markers and anti-cancer agents. Our preliminary analysis revealed the existence of a single D-fucose binding lectin from the grub serum of the darkling beetle, Z. morio. Here, whole transcriptome analysis using entire gut and fat body tissues of grub of Z. morio with Illumina NovaSeq6000 sequencing platform revealed the existence of a novel fucose binding lectin (ZmFBL) displaying significant similarity to Diaphorina citri fucolectin-3 and possessing fucolectin-tachylectin4-pentraxin domain. Molecular docking between the structure of the predicted FBL transcript from Z. morio and D-fucose demonstrated the lowest binding energy of −5.4 kcal/mol. The MD simulation and MM/PBSA analysis furnished insights into the binding stability of D-fucose and the ZmFBL. A similar transcript was also identified from the fat body transcriptome. A 2.49-fold increase in the ZmFBL expression was observed in the fat body than the whole gut as evidenced from the relative quantitation using RT-PCR.

MCT1 lactate transporter blockade re-invigorates anti-tumor immunity through metabolic rewiring of dendritic cells in melanoma

Dendritic cells (DC) are key players in antitumor immune responses. Tumors exploit their plasticity to escape immune control; their aberrant surface carbohydrate patterns (e.g., glycans) shape immune responses through lectin binding, and manipulate the metabolism of immune cells, including DCs to alter their function and escape immune surveillance. DC metabolic reprogramming could induce immune subversion and tumor immune escape. Here we explore metabolic features of human DC subsets (cDC2s, cDC1s, pDCs) in melanoma, at single cell level, using the flow cytometry-based SCENITH (Single-Cell ENergetIc metabolism by profiling Translation inHibition) method. We demonstrate that circulating and tumor-infiltrating DC subsets from melanoma patients are characterized by altered metabolism, which is linked to their activation status and profile of immune checkpoint expression. This altered metabolism influences their function and affects patient clinical outcome. Notably, melanoma tumor cells directly remodel the metabolic profile of DC subsets, in a glycan-dependent manner. Strikingly, modulation of the mTOR/AMPK-dependent metabolic pathways and/or the MCT1 lactate transporter rescue cDC2s and cDC1s from skewing by tumor-derived glycans, Sialyl-Tn antigen and Fucose, and restore anti-tumor T-cell fitness. Our findings thus open the way for appropriate tuning of metabolic pathways to rescue DCs from tumor hijacking and restore potent antitumor responses.

Endothelial characteristics of cardiac stem cell antigen-1 expressing cells and their relevance to right ventricular adaptation.

A growing body of evidence suggest that the stem cell antigen-1 expressing (Sca-1+) cells in the heart may be the cardiac endothelial stem/progenitor cells. Their endothelial cell (EC) functions, and their role in RV physiology and pathophysiology of right heart failure (RHF) remains poorly defined. This study investigated EC characteristics of rat cardiac Sca-1+ cells, assessed spatial distribution and studied changes in Sca1+ cells during RV remodelling in monocrotaline (MCT) model of pulmonary hypertension and RV remodeling. First, flow cytometry analysis of adult male and female Sprague Dawley (SD) and Fischer CDF rat heart cells was performed, and we observed that the majority of Sca-1+ cells also expressed CD31, an EC marker. Furthermore, Sca-1+ cells showed acetylated low-density lipoprotein (ac-LDL) uptake and lectin binding similar to CD31+ cells from the same heart. The Sca-1+ cells also demonstrated network formation when plated on Matrigel. In the MCT treated rats, we observed increase in RV hypertrophy that correlated with the reduction in the abundance of Sca-1+CD31+ cells in the RV. Together, the cardiac Sca-1+ cells in the heart are endothelial stem/progenitor-like cells. These cells have higher abundance in the RV and may play a role in RV adaptation.

A pilot trial for efficacy confirmation of 6′-sialyllactose supplementation in GNE myopathy: Randomized, placebo-controlled trial

GNE myopathy is a rare genetic muscle disorder characterized by initial ankle dorsiflexor weakness and the presence of rimmed vacuoles in muscle histopathology. Biallelic mutations in the GNE gene are causative, leading to reduced production of sialic acid. In our previous clinical trial, we used 6′-sialyllactose (6SL) as a supplement to increase sialic acid levels and compared the effects of 6SL at doses of 3 g and 6 g. The findings from the trial revealed superior outcomes in muscle strength, attenuation of muscle degeneration, and bioavailability in the 6 g group.This trial was planned to complement the lack of placebo arm from the previous trial and to provide more conclusive evidence for therapeutic value of 6SL in GNE myopathy. Of the 11 participants, five were allocated to the 6SL and six to the placebo group after undergoing 12 weeks of pre-study observation and stratified randomization. At every visit with an interval of 12 weeks for 48 weeks, all participants underwent muscle strength measurement, muscle MRI, biochemical evaluations, 6-min-walk test, and completed a questionnaire.No safety concerns arose during the trial period. Muscle strength, excluding hand grip power, did not show a significant difference between the two groups, which is attributed to the lack of pronounced muscle strength decline in both groups. Hand grip power tended to decrease in both groups, and this decline was statistically significant in the placebo group (p = 0.0004). The fat fraction measured by MRI showed the most significant results in the posterior thigh. The increase in fat fraction, indicating muscle degeneration, was statistically significant between the two groups (p = 0.0004). Although no statistically significant differences were observed between the groups in anterior thigh and both anterior and posterior lower leg, a trend of slowed increase in fat fraction was noted in the 6SL group compared to the placebo group starting from 24 or 36 weeks. Resialylation of cell surface glycoconjugate was demonstrated in 6SL group by measuring lectin bindings on peripheral blood monocytes.The GNEM-FAS, used to assess patient-reported outcomes, did not show statistical significance in the total score or any of the three domains. However, the tendency for scores in the self-care and upper extremity domains to rebound after 24 weeks in the 6SL group suggests the potential for long-term benefits.The effect of 6SL on muscle strength appeared to be minimal compared to our previous clinical trial, likely due to the short duration of the study and the inclusion of relatively early-stage patients. However, the changes in fat fraction measured by muscle MRI and the results of biochemical assays are more promising, suggesting potential benefits with long-term administration in the future.

A Serum D-Fucose Binding Lectin With B Cell Mitogenic Activity From the Grub of the Darkling Beetle Zophobas morio.

Lectins that can recognize and bind to carbohydrates and glycoconjugates are at the epicentre of research owing to their prospective applications. In the present study, a D-fucose binding lectin from the serum of darkling beetle, Zophobas morio was purified and their mitogenic potential over human B-cells was evaluated. Biochemical assays on the preliminary characterization revealed the occurrence of single D-fucose binding lectin. Through single step affinity chromatography using D-fucose coupled Epoxy-activated Sepharose 6B, lectin (ZmFBL) with a molecular mass of ~192 kDa from the serum of Z. morio was purified with homogeneity. The HA activity of the purified ZmFBL remained stable between the pH 7 and 12 and was thermo-tolerant up to a temperature of 40°C. MALDI-TOF-MS analysis of native lectin disclosed fucose-binding nature of the ZmFBL with mitogenic property. The results of functional analysis of purified ZmFBL on the effect on B-cell proliferation revealed that ZmFBL at the concentrations of 31.25 μg and 62.50 were the ideal concentrations that significantly enhanced (approximately 2.5-fold over control) the proliferation of the B-lymphocyte population up to 72 h of treatment without any cytotoxicity. The outcome of this study could possibly prove beneficial in the investigation on the potential use of ZmFBL as immunostimulant and in immunosuppressive treatments.

Glycosylation in Canine Placentas.

This article describes the carbohydrate composition of early and mature placentas from bitches, detected by lectin histochemistry. Formalin-fixed placental samples from 11 mixed-breed bitches have been assigned to the 'early' or the 'mature' placenta group, processed by the routine histological technique and labelled with a panel of 14 biotinylated lectins. The glycan distribution was almost completely preserved over pregnancy. Cytotrophoblast cells expressed high-mannose N-linked residues and N-acetyl-D-glucosamine, whereas mannose N-linked residues, N-acetyl-D-glucosamine and β- and α-D-galactose/N-acetyl-D-galactosamine have been found in the syncytiotrophoblast. The maternal and foetal endothelial binding pattern was enriched in β-D-galactose, α-D-mannose in non-bisected bi/tri-antennary N-linked complex and Galα1,3Gal-Galα1,4Gal. Both deep and superficial glands showed a great variety of glycoconjugates, such as D-mannose, D-glucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, galactose, N-acetylneuraminic acid and fucose. Most results in this study were consistent with those previously reported in canine and feline mature placentas; here, early placentas have been analysed and the lectin binding pattern of mature placentas has been further described. These studies on canine placentas contribute to detecting and understanding glycome changes in pathophysiological conditions.

Convergence of plant sterols and host eukaryotic cell-derived defensive lipids at the infectious pathogen-host interface.

Plant sterols (PSs) exhibit intrinsic functions such as antibacterial effects. Their effects simultaneously on both host-mediated and bacteria-mediated pathogenesis are not yet fully understood. We hypothesized that when absorptive cells, defensive cells and detoxer cells are cultured together, their convergent response to an infectious pathogen depends on the molecular mimicry between the ingested sterols and their own defensive lipids. A human triple cell co-culture model incorporating colonocytes, macrophages, and hepatocytes was established. Cocultures were stimulated with Klebsiella pneumoniae 52145 (Kp52145) in the presence of pure plant sterol (β-sitosterol, PS) for 6h. Changes in the structural health markers of the stimulated cocultured cells and their immune response and biochemical markers of pathogenicity were determined. PS significantly inhibited the secretion of cytokines induced by Kp52145. Cell viability was higher in the Kp52145+PS group compared to the Kp52145 alone group. PS decreased Kp52145-induced marker of pathogenicity (SOD), accompanied by reduced levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), mannose binding lectin (MBL), and pentraxin 3 (PTX3) which are the mediators and enzymes associated with the inflammatory response to an infectious-inflamed milieu. PS recovered Kp52145-decreased peroxidase (POX), catalase (CAT), complement component 3 (C3), and high-density lipoprotein cholesterol (HDL-C) values. Convergence of ingested plant sterols and host eukaryotic cell-derived defensive lipids mitigates the disruptive effects of bacterial toxic effector molecules. Structural or immunological similarities (molecular mimicry) between ingested plant sterols and host defensive lipids play an important role in modulating bacterial signalling that occurs at the pathogen-host interface and in the mitigation of infection- and inflammation-driven pathological processes.

Sialylation inhibition improves macrophage mediated tumor cell phagocytosis of breast cancer cells triggered by therapeutic antibodies of different isotypes.

Tumor cell phagocytosis by macrophages is considered a relevant mechanism of action for many therapeutic IgG antibodies. However, tumor cells employ several mechanisms to evade immune recognition, including hypersialylation. Here, we describe how reduction of sialic acid exposure on tumor cells promotes antibody-dependent tumor cell phagocytosis (ADCP) by macrophages. Incubation with the sialyltransferase inhibitor (STi) P-3Fax-Neu5Ac reduced sialylation on two breast cancer cell lines, rendering these cells more susceptible to macrophage mediated phagocytosis by EGFR or HER2 antibodies. This was observed with not only IgG1 and IgG2 antibodies but also IgA2 variants. These results show that inhibiting sialic acid exposure triggers enhanced tumor cell phagocytosis by macrophages irrespective of the antibody isotype and the tumor target antigen. Investigating the underlying mechanisms of enhanced ADCP, we observed reduced binding of soluble sialic acid-binding immunoglobulin-like lectins (Siglec)-7 and Siglec-9 to tumor cells after sialylation inhibition. However, Fc silent blocking antibodies against Siglec-7 or Siglec-9, or their combination, only marginally improved ADCP. Our results further promote the concept of cancer hypersialylation as immune escape mechanism, which could serve as target to improve tumor immunotherapy with monoclonal antibodies.

Analysis of the binding specificity of N‐acetylgalactosamine (GalNAc)‐binding lectin isolated from Kappaphycus alvarezii to SARS‐CoV‐2 sugar moieties

SUMMARYN‐acetylgalactosamine (GalNAc)‐binding lectin is one of the beneficial lectins because it has the ability to bind to glycoproteins or glycolipids containing GalNAc. Its potential applications include use as a drug delivery system, as well as for diagnostic tools and therapeutic interventions. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike proteins are known to be heavily glycosylated, and GalNAc‐binding lectin can potentially bind with these glycoproteins. The present study specifically investigated the interactions between GalNAc‐binding lectin from Kappaphycus alvarezii (Rhodophyta) and the sugars and glycoproteins present on the S protein of the SARS‐CoV‐2 virus. The GalNAc‐specific lectin was successfully isolated from K. alvarezii var. tangan‐tangan (Loving Beauty) and purified using a Sepharose 6B‐GalNAc affinity column. This lectin demonstrated stability within the temperature range 30–80°C and pH range 3–9. The minimum inhibitory concentration of this lectin was determined to be 0.001 mg mL−1. Sugar specificity tests confirmed its ability to bind to the sugars and glycoproteins reported to be present on the S protein of the SARS‐CoV‐2 virus envelope. These results suggest that the potential of GalNAc‐specific lectin from K. alvarezii can be further explored, making it a promising candidate for therapeutic interventions against coronavirus.

O-glycosylation is essential for cell surface expression of the transcobalamin receptor CD320

CD320 is a cell surface receptor that mediates vitamin B12 uptake in most tissues. To date, the mechanisms that regulate CD320 expression on the cell surface are not fully understood. In this work, we studied CD320 expression in transfected human embryonic kidney (HEK) 293 and hepatoma HepG2 cells. By glycosidase and trypsin digestion, monensin and brefeldin treatment, western blotting, flow cytometry, and lectin binding, we found that CD320 underwent N- and O-glycosylation and sialylation, resulting in a ∼70-kDa band that formed a high-molecular-weight complex on the cell surface. Site-directed mutagenesis altering Asn126, Asn195, and Asn213 residues, individually or together, abolished N-glycosylation in CD320 but did not block its intracellular trafficking and expression on the cell surface in HEK293 and HepG2 cells. In contrast, treatment of the cells with Ben-gal, a structural analog of GalNAc-α-1-O-Ser/Thr, inhibited O-glycosylation and cell surface expression of CD320 and decreased vitamin B12 uptake. Analysis of CD320 deletion mutants indicated that O-glycosylation sites in a Ser/Thr-rich region near the transmembrane domain were important for CD320 expression on the cell surface. These results reveal an important role of O-glycans, but not N-glycans, in the intracellular trafficking and cell surface expression of CD320, providing new insights into the cellular mechanisms in regulating CD320 function and vitamin B12 metabolism.

Selective Glycan Presentation in Liquid‐Ordered or ‐Disordered Membrane Phases and its Effect on Lectin Binding

AbstractGlycan‐protein interactions play a key role in various biological processes from fertilization to infections. Many of these interactions take place at the glycocalyx—a heavily glycosylated layer at the cell surface. Despite its significance, studying the glycocalyx remains challenging due to its complex, dynamic, and heterogeneous nature. This study introduces a glycocalyx model allowing for the first time to control spatial organization and heterogeneity of the glycan moieties. Glycan‐mimetics with lipid‐moieties that partition into either liquid‐ordered (Lo, lipid rafts) or liquid‐disordered (Ld) phases of giant unilamellar vesicles (GUVs), which serve as simplified cell membrane models mimicking lipid rafts, are developed. This phase‐specific allocation allows controlled placement of glycan motifs in distinct membrane environments, creating heteromultivalent systems that replicate the natural glycocalyx's complexity. We show that phase localization of glycan mimetics significantly influences recruitment of protein receptors to the membrane. Glycan‐conjugates in the ordered phase demonstrate enhanced lectin binding, supporting the idea that raft‐like domains facilitate stronger receptor interactions. This study provides a platform for systematically investigating spatial and dynamic presentation of glycans in biological systems and presents the first experimental evidence that glycan accumulation in lipid rafts enhances receptor binding affinity, offering deeper insights into the glycocalyx‘s functional mechanisms.

Selective Glycan Presentation in Liquid-Ordered or -Disordered Membrane Phases and its Effect on Lectin Binding.

Glycan-protein interactions play a key role in various biological processes from fertilization to infections. Many of these interactions take place at the glycocalyx-a heavily glycosylated layer at the cell surface. Despite its significance, studying the glycocalyx remains challenging due to its complex, dynamic, and heterogeneous nature. This study introduces a glycocalyx model allowing for the first time to control spatial organization and heterogeneity of the glycan moieties. Glycan-mimetics with lipid-moieties that partition into either liquid-ordered (Lo, lipid rafts) or liquid-disordered (Ld) phases of giant unilamellar vesicles (GUVs), which serve as simplified cell membrane models mimicking lipid rafts, are developed. This phase-specific allocation allows controlled placement of glycan motifs in distinct membrane environments, creating heteromultivalent systems that replicate the natural glycocalyx's complexity. We show that phase localization of glycan mimetics significantly influences recruitment of protein receptors to the membrane. Glycan-conjugates in the ordered phase demonstrate enhanced lectin binding, supporting the idea that raft-like domains facilitate stronger receptor interactions. This study provides a platform for systematically investigating spatial and dynamic presentation of glycans in biological systems and presents the first experimental evidence that glycan accumulation in lipid rafts enhances receptor binding affinity, offering deeper insights into the glycocalyx's functional mechanisms.

Effect of GB1107, a novel galectin-3 inhibitor on pro-fibrotic signalling in the liver

Background and purposeGalectin-3 (Gal-3) is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. GB1107 is a novel orally active Gal-3 small molecule inhibitor that has high affinity for Gal-3 >1000-fold selectively over other galectins. The aim of this study was to characterise GB1107 and galectin-3 in vitro and in vivo in the context of fibrosis signalling and liver disease. Experimental approachLiver fibrosis was induced by administration of CCl4 twice weekly by intraperitoneal injection in mice for 8 weeks. GB1107 was orally administered once daily (10 mg/kg) for the last 4 weeks of CCl4 treatment. Fibrosis was assessed by picrosirius red staining of FFPE sections. Liver enzymes, Gal-3 and downstream biomarkers were assessed in liver and plasma. Paired-end sequencing was performed on the Nextseq 2000 platform. Pathway enrichment analysis was performed to determine enrichment of differentially expressed genes (DEGs) within Reactome pathways and Gene Ontology (GO) terms. Key resultsGB1107 significantly reduced plasma transaminases and liver Gal-3 and reduced liver fibrosis. RNAseq analysis of whole liver showed that 1659 DEGs were identified with CCl4 treatment compared to control. Pathways enriched in up-regulated genes in the CCl4 group included those related to the extracellular matrix, collagen biosynthesis and assembly, cell cycle and the immune system. Comparing GB1107 treatment with CCl4 control 1147 DEGs were identified. GB1107 effectively reversed the majority of the CCl4 induced gene changes. Conclusions and implicationsGB1107 attenuated liver fibrosis and highlights Gal-3 as a therapeutic target for hepatic fibrosis.

Targeting the Leloir Pathway with Galactose-Based Antimetabolites in Glioblastoma.

Glioblastoma (GBM) uses Glut3 and/or Glut14 and the Leloir pathway to catabolize D-Galactose (Gal). UDP-4-deoxy-4-fluorogalactose (UDP-4DFG) is a potent inhibitor of the two key enzymes, UDP-galactose-4-epimerase (GALE) and UDP-Glucose 6-dehydrogenase (UGDH), involved in Gal metabolism and in glycan synthesis. The Gal antimetabolite 4-deoxy-4-fluorogalactose (4DFG) is a good substrate for Glut3/Glut14 and acts as a potent glioma chemotherapeutic. Primary GBM cell cultures were used to examine toxicity and alterations in glycan composition via lectin binding in fixed cells and by Western blots. Toxicity/efficacy in vivo data was performed in mouse flank and intracranial models. The effect of 4DFG on D-glucose (Glc) metabolism in GBM cells was assessed by using 13C NMR-based tracer studies. 4DFG is moderately potent against GBM cells (IC50: 125-300 µM). GBM glycosylation is disrupted by 4DFG. Survival analysis in an intracranial mouse model showed that treatment with 4DFG (6 × 25 mg/kg of 4DFG, intravenously) improved outcomes by three-fold (p < 0.01). Metabolic flux analysis revealed that both glycolytic and mitochondrial metabolic fluxes of [U-13C]Glc were significantly decreased in the presence of 4DFG in GBM cells. A functional Gal-scavenging pathway in GBM allows Gal-based antimetabolites to act as chemotherapeutics. 4DFG is metabolized by GBM in vitro and in vivo, is lethal to GBM tumors, and is well tolerated in mice.

Sugar-binding Profiles of the Mesothelial Glycocalyx in Frozen Tissues of Mice Revealed by Lectin Staining

The mesothelium is a non-adhesive protective surface that lines the serosal cavities and organs within the body. The glycocalyx is a complex structure that coats the outer layer of the mesothelium. However, due to the limitations of conventional fixation techniques, studies on glycans are limited. In this study, lectin staining of frozen tissues was performed to investigate the diversity of glycans in the glycocalyx of mesothelial cells in mice. Datura stramonium lectin (DSL), which recognizes lactosamine and binds to Galectin-3 and -1, was broadly bound to the mesothelial cells of the visceral and parietal peritoneum but not to the pancreas, liver, intestine, or heart. Furthermore, human mesothelial cells in the omentum and parietal peritoneum were positive for DSL. Erythrina cristagalli lectin binding was specific to mesothelial cells in the parietal peritoneum, that is, the pleura, diaphragm, and peritoneum. Intriguingly, surface sialylation, the key element in reducing peritoneal dissemination and implantation, and promoting ascites formation by ovarian carcinoma cells, was much higher in the parietal peritoneum than in the omentum. These findings revealed slight differences in the glycans of mesothelial cells of different organs, which may be related to clinical diseases. These results also suggest that there may be differences in the functions of parietal and visceral mesothelial cells.

Accelerated wound healing and reduced scar formation induced by D-mannose: a possible role of mannose binding lectin

Wounds are most commonly caused by accidents, surgery and burns, and can be internal or external. Naturally, the wound healing process can take a long time and lead to scarring. In this study we present a technique to shorten wound healing time and prevent or mitigate scarring using D-mannose that is applied directly on the wound. The results showed that the healing time is almost halved compared to treatment with povidone-iodine solution which is an antiseptic widely used in surgery. D-Mannose is neither an antisepsis nor an antibiotic. We propose a posssible mechanism by which D-mannose binds to D-mannose binding lectin and immediately activates the innate immune system that ultimately phagocytizes pathogens and clears the wound of degraded cells and materials, which reduces inflammation and implicitly wound healing time. D-Mannose also intervenes in the coagulation process by binding to fibrinogen, generating a finer and denser fibrin, which visibly reduces collagen scars. Our findings show that applying D-mannose directly on the wound as a powder shortens wound healing time and visibly attenuates scarring. Apart from the unaesthetic appearance, these scars can also cause a certain tissue dysfunction, regardless of the affected organ.Graphical abstract

Strategies beyond Decellularization for Optimal Tissue Engineering of Cardiac Xenografts

The immunogenic carbohydrates, known as xenoantigens such as the Galα1-3Gal (α-Gal) epitope, and the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), play a major role in the immune response to xenotransplantation. Optimal decellularization protocols and strategies beyond decellularization are essential to eliminate xenoimmunogenicity. The aim of this study was to evaluate the safety and efficasy of our novel decellularization protocol and carbohydrase such as Peptide N-Glycosidase F (PNGase-F) for the removal of xenoantigens. We investigated the biomechanical properties, and efficacy for xenoantigen removal through expression of carbohydrate-binding lectins in porcine pericardium decellularized and treated with PNGase-F. Our novel decellularization protocols using 0.25% SDS + Triton X-100 through the multi-step methods with hypotonic, isotonic and hypertonic buffer solution demonstrated complete decellularization without histological changes. There were no histological changes depending on PNGase-F treatment concentration (0~2 unit/ml). There were no significant differences in tensile stress, tensile displacement, tensile strain at break, and permeability tests among porcine pericardia treated with different concentrations of PNGase-F. PNGase-F-treated porcine pericardium was stained with Jacalin, MAL I, WGA, RCA, GSL, ECA, PNA, SBA, WFA, and DSL, and showed lower fluorescence than native pericardium. PNGase-F treatment also resulted in a significant inhibition of lectin binding levels in a concentration-dependent manner. The fluorescent signal of decellularization effectively decreased lectin expression. Additional PNGase-F treatment for decellularization significantly reduced lectin expression without differences in PNGase-F concentration, demonstrating the synergistic effect of decellularization and PNGase-F. In conclusion, our novel decellularization protocols combined with PNGase-F treatment effectively removed the carbohydrates-associated xenoimmunogenicity and maintained biomechanical stability for cardiac xenografts. Our strategies beyond decellularization for optimal tissue engineering of cardiac xenografts can improve the biocompatibility of the graft.

Sub-10 μm Soft Interlayers Integrating Patterned Multivalent Biomolecular Binding Environments.

Designing surfaces that enable controlled presentation of multivalent ligand clusters (e.g., for rapid screening of biomolecular binding constants or design of artificial extracellular matrices) is a cross-cutting challenge in materials and interfacial chemistry. Existing approaches frequently rely on complex building blocks or scaffolds and are often specific to individual substrate chemistries. Thus, an interlayer chemistry that enabled efficient nanometer-scale patterning on a transferrable layer and subsequent integration with other classes of materials could substantially broaden the scope of surfaces available for sensors and wearable electronics. Recently, we have shown that it is possible to assemble nanometer-resolution chemical patterns on substrates including graphite, use diacetylene polymerization to lock the molecular pattern together, and then covalently transfer the pattern to amorphous materials (e.g., polydimethylsiloxane, PDMS), which would not natively enable high degrees of control over ligand presentation. Here, we develop a low-viscosity PDMS formulation that generates very thin films (<10 μm) with dense cross-linking, enabling high-efficiency surface functionalization with polydiacetylene arrays displaying carbohydrates and other functional groups (up to 10-fold greater than other soft materials we have used previously) on very thin films that can be integrated with other materials (e.g., glass and soft materials) to enable a highly controlled multivalent ligand display. We use swelling and other characterization methods to relate surface functionalization efficiency to the average distance between cross-links in the PDMS, developing design principles that can be used to create even thinner transfer layers. In the context of this work, we apply this approach using precision glycopolymers presenting structured arrays of N-acetyl glucosamine ligands for lectin binding assays. More broadly, this interlayer approach lays groundwork for designing surface layers for the presentation of ligand clusters on soft materials for applications including wearable electronics and artificial extracellular matrix.

Phylogenetic variations in a novel family of hyperstable apple snail egg proteins: insights into structural stability and functional trends.

The relationship between protein stability and functional evolution is little explored in proteins purified from natural sources. Here, we investigated a novel family of egg proteins (Perivitellin-1, PV1) from Pomacea snails. Their remarkable stability and clade-related functions in most derived clades (Canaliculata and Bridgesii) make them excellent candidates for exploring this issue. To that aim, we studied PV1 (PpaPV1) from the most basal lineage, Flagellata. PpaPV1 displays unparalleled structural and kinetic stability, surpassing PV1s from derived clades, ranking among the most hyperstable proteins documented in nature. Its spectral features contribute to a pale egg coloration, exhibiting a milder glycan binding lectin activity with a narrower specificity than PV1s from the closely related Bridgesii clade. These findings provide evidence for substantial structural and functional changes throughout the genus' PV1 evolution. We observed that structural and kinetic stability decreased in a clade-related fashion and was associated with large variations in defensive traits. For instance, pale PpaPV1 lectin turns potent in the Bridgesii clade, adversely affecting gut morphology, while giving rise to brightly colored PV1s providing eggs with a conspicuous, probably warning signal in the Canaliculata clade. This work provides a comprehensive comparative analysis of PV1s from various apple snail species within a phylogenetic framework, offering insights into the interplay among their structural features, stability profiles and functional roles. More broadly, our work provides one of the first examples from natural evolution showing the crucial link among protein structure, stability and evolution of new functions.

The galectin-3 inhibitor selvigaltin reduces liver inflammation and fibrosis in a high fat diet rabbit model of metabolic-associated steatohepatitis.

Galectin-3 is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. Selvigaltin (previously known as GB1211) is a novel orally active galectin-3 small molecule inhibitor that has high affinity for galectin-3 (human KD = 25nM; rabbit KD = 12nM) and high oral bioavailability in rabbits and man. In this study the efficacy of selvigaltin was investigated in a high fat diet (HFD) rabbit model of metabolic-associated steatohepatitis (MASH). Male New Zealand White rabbits were individually caged under standard conditions in a temperature and humidity-controlled room on a 12h light/darkness cycle. After 1week of regular diet (RD), rabbits were randomly assigned for 8 or 12weeks to different groups: RD/vehicle, RD/selvigaltin, HFD (8weeks), HFD/vehicle and HFD/selvigaltin (0.3, 1.0, 5.0 or 30mg/kg selvigaltin with vehicle/selvigaltin p.o. dosed therapeutically q.d. 5days per week from week 9 or 12). Liver inflammation, steatosis, ballooning, and fibrosis was measured via blood metabolic markers, histomorphological evaluation [Oil Red O, Giemsa, Masson's trichome, picrosirius red (PSR) and second harmonic generation (SHG)], and mRNA and protein expression. Steatosis, inflammation, ballooning, and fibrosis were all increased from RD to HFD/vehicle groups. Selvigaltin demonstrated target engagement by significantly decreasing galectin-3 levels in the liver as measured via immunohistochemistry and mRNA analysis. Selvigaltin dose-dependently reduced biomarkers of liver function (AST, ALT, bilirubin), inflammation (cells foci), and fibrosis (PSR, SHG), as well as decreasing the mRNA and protein expression of several key inflammation and fibrosis biomarkers (e.g., IL6, TGFβ3, SNAI2, collagen). Doses of 1.0 or 5.0mg/kg demonstrated consistent efficacy across most biological endpoints supporting the current clinical doses of selvigaltin being investigated in liver disease. Selvigaltin significantly reduced hepatic inflammation and fibrosis in an HFD rabbit model of MASH following therapeutic dosing for 4 weeks in a dose-dependent manner. These data support the human selvigaltin dose of 100mg b.i.d. that has been shown to reduce key liver biomarkers during a clinical study in liver cirrhosis.