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Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus-particularly the risk allele-drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13.

Post-traumatic osteoarthritis (PTOA) is a painful joint disease characterized by the degradation of bone, cartilage, and other connective tissues in the joint. PTOA is initiated by trauma to joint-stabilizing tissues, such as the anterior cruciate ligament, medial meniscus, or by intra-articular fractures. In humans, ~50% of joint injuries progress to PTOA, while the rest spontaneously resolve. To better understand molecular programs contributing to PTOA development or resolution, we examined injury-induced fluctuations in immune cell populations and transcriptional shifts by single-cell RNA sequencing of synovial joints in PTOA-susceptible C57BL/6J (B6) and PTOA-resistant MRL/MpJ (MRL) mice. We identified significant differences in monocyte and macrophage subpopulations between MRL and B6 joints. A potent myeloid-driven anti-inflammatory response was observed in MRL injured joints that significantly contrasted the pro-inflammatory signaling seen in B6 joints. Multiple CD206+ macrophage populations classically described as M2 were found enriched in MRL injured joints. These CD206+ macrophages also robustly expressed Trem2, a receptor involved in inflammation and myeloid cell activation. These data suggest that the PTOA resistant MRL mouse strain displays an enhanced capacity of clearing debris and apoptotic cells induced by inflammation after injury due to an increase in activated M2 macrophages within the synovial tissue and joint space.

Current evidence suggests that ontogeny may account for the functional heterogeneity of some tissue macrophages, but not others. Here, we asked whether developmental origin drives different functions of skin Langerhans cells (LCs), an embryo-derived mononuclear phagocyte with features of both tissue macrophages and dendritic cells. Using time-course analyses, bone marrow chimeras, and fate tracing models, we found that the complete elimination of embryo-derived LCs at steady state results in their repopulation from circulating monocytes. However, monocyte-derived LCs inefficiently replenished the epidermal niche. Instead, these cells preferentially migrated to skin-draining lymph nodes. Mechanistically, we show that the enhanced migratory capability of monocyte-derived LCs is associated with higher expression of CD207/Langerin, a C-type lectin involved in the capture of skin microbes. Our data demonstrate that ontogeny plays a role in the migratory behavior of epidermal LCs.

BACKGROUND: Worsening renal function (WRF) is common in hospitalized patients being treated for acute heart failure. However, discriminating clinically significant WRF remains challenging. In patients hospitalized with acute heart failure, we evaluated if blood and urine biomarkers of cardiac and kidney dysfunction were associated with adverse outcomes. METHODS: We identified 175 of 927 participants in the AKINESIS study (Acute Kidney Neutrophil Gelatinase-Associated Lipocalin Evaluation of Symptomatic Heart Failure Study) who met criteria for stage 1 or 2 Kidney Disease: Improvement Global Outcomes acute kidney injury during the first 3 days of hospitalization. We measured 24 blood and urine biomarkers from specimens collected within 24 hours of meeting acute kidney injury criteria. The primary composite outcome consisted of worsening WRF (higher acute kidney injury stage), need for dialysis, or death at 30 days. Biomarkers association with the composite outcome was assessed with logistic regression by tertiles and area under the curve (AUC). RESULTS: Of the 175 participants, 32 (18%) developed the primary composite outcome. Only history of chronic kidney disease was significantly different between those with and without the composite outcome. The highest tertile of plasma Gal-3 (galectin-3) and urine epidermal growth factor were associated with increased odds of the composite outcome compared with the lowest tertile in unadjusted analyses. After adjusting for serum creatinine, systolic blood pressure, and blood urea nitrogen, only the highest tertile of Gal-3 was associated with greater odds of the composite outcome (odds ratio, 4.6 [95% CI, 1.4-16.0). Gal-3 had the highest AUC (0.70 [95% CI, 0.58-0.82]), while epidermal growth factor had a lower AUC (0.63 [95% CI, 0.53-0.74]). Notably, urine biomarkers of kidney tubule injury were not associated with the composite outcome. CONCLUSIONS: Tubular injury does not occur in most patients with acute heart failure experiencing WRF, consistent with the functional mechanisms of WRF in this patient population. REGISTRATION: URL: https://www.clinicaltrials.gov/study/NCT01291836?term=NCT01291836&rank=1; Unique identifier: NCT01291836.

Prostate cancer is the second leading cause of male cancer death in the U.S. Current immune checkpoint inhibitor-based immunotherapies have improved survival for many malignancies; however, they have failed to prolong survival for prostate cancer. Siglecs (sialic acid-binding immunoglobulin-like lectins) are expressed on immune cells and regulate their function. Siglec-7 and Siglec-9 contribute to immune evasion in cancer by interacting with sialic acid-containing glycoprotein ligands on cancer cells. However, the role of Siglec-7/9 receptors and their ligands in prostate cancer remains poorly understood. Here, we find that Siglec-7 and Siglec-9 are associated with poor prognosis in patients with prostate cancer and are highly expressed in myeloid cells, including macrophages, in prostate tumor tissues. Siglec-7 and -9 ligands were expressed in prostate cancer cells and human prostate tumor tissues. Blocking the interactions between Siglec-7/9 and sialic acids inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice in vivo. Using a CRISPRi screen and mass spectrometry, we identified CD59 as a candidate Siglec-9 ligand in prostate cancer. The identification of Siglec-7 and -9 as potential therapeutic targets, including the CD59/Siglec-9 axis, opens up opportunities for immune-based interventions in prostate cancer.

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate-binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a ΔlecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the ΔlecF/X ECM could explain the double mutants hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment.

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, the neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing their refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that the reduction in repulsive net charge of the purified, recombinant reflectin-either (for the first time) by generalized anionic screening with salt or by pH titration-drives a finely tuned, precisely calibrated increase in the size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. The precise proportionality between the assembly size and charge neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth by a continual, equilibrium tuning of the balance between the proteins Coulombic repulsion and short-range interactive forces. The resulting stability of reflectin assemblies with time ensures a reciprocally precise control of the particle number concentration, encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The charge regulation of reflectin assembly precisely fine-tunes a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

Patients with decreased levels of CD18 (β2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMβ2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the β-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the outside-in signaling for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1β release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.

UNLABELLED: Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using in vivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.

Lectins are proteins that specifically recognize carbohydrates on cell membranes, triggering several cellular events such as apoptosis of cancer-transformed cells; however, the mechanisms of action remain incompletely understood. Our research group has reported that a concentrated fraction of Tepary bean lectins (Phaseolus acutifolius; TBLF) exhibits the concentration-dependent induction of apoptosis in colon cancer cells by caspase activation. It is well established that an increase in cytoplasmic calcium ([Ca2+]i) initiates intracellular signals involved in processes such as exocytosis, gene transcription, apoptosis, cell cycle regulation, and muscle contraction, among others. Furthermore, dysregulated calcium signaling has been implicated in various diseases, including certain neurological disorders and cancer. In this study, we aim to demonstrate the effects of native TBLF lectins and a recombinant lectin (rTBL-1) on calcium mobility in breast cancer cells (MCF-7) and non-cancerous cells (MCF-12F). Both TBLF and rTBL-1 increased intracellular calcium concentrations and mobilized calcium from intracellular stores in a concentration-dependent manner; however, the two cell lines exhibited differential responses. While MCF-12F cells restored cytoplasmic calcium concentration, MCF-7 cells maintained a high intracellular calcium concentration. This strongly suggests that lectins can elicit differential cellular responses in cancer and non-cancer cells due to variations in their intrinsic mechanisms of calcium homeostasis. Finally, we demonstrated that TBLF and rTBL-1 can differentially alter Metabolic Cellular Activity (MCA) as a direct measure of cell viability (CVi) in both cell lines. These findings strengthen the evidence of the therapeutic potential of Tepary bean lectins. Undoubtedly, further studies will be necessary to elucidate the mechanisms underlying their applications. [ABSTRACT FROM AUTHOR]

The prevalence of cancer deaths globally and domestically is higher especially due to the deferment of diagnosis and lack of facilities for women's reproductive cancers. The present review focussed to explore the application of lectins in cancer theranostics. Though there is cancer diagnostic and treatment available there is no promising early diagnostic tool and effective treatment available for the cancer which is the major concern. Lectins are cellulose-binding proteins that are strongly determined in saccharide groups of glycans, glycopeptides, or glycolipids. In the concomitance of events in cells, carbohydrates, and proteins, lectins play an important role. Lectins bind superiorly to the cancer cell membrane and their receptors induce the cytotoxic effect, which results in caspase-mediated cell death, and prohibits tumour development. Lectin snuffing also reveals polyamine stocks and impedes the growth of cancerous cells. They affect the cell cycle by non-apoptotic aggregation, seizure of the cell cycle phase G2, M, and the mediation of caspases. It can also adversely affect the action of telomerase and hinder vascularisation. They promote immunomodulation and adversely limit protein synthesis. Their easy availability and its characteristics support its use in cancer diagnosis and therapy, despite their small corollary effects. Future investigations recommend focussing more on the key applications of lectin by reducing its concurrent effects and carrying out more in-vitro investigations. However, the use of lectin formulations for cancer theranostics is a new area in cancer detection and treatment. In this review, plant lectin appears to be a potential target for cancer research in the fields of diagnosis and theranostics. [ABSTRACT FROM AUTHOR]

Background: Shell and pearl formation in bivalves is a sophisticated biomineralization process that encompasses immunological and mineralization aspects, particularly during shell repair and the initial stages of pearl cultivation when a nucleus is inserted. Here, we describe a novel C-type lectin, HcLec1, isolated and characterized from the freshwater pearl mussel Hyriopsis cumingii Lea. Methods: Immune challenge, RNA interference (RNAi) experiments, ELISA, and antibacterial assays were employed to investigate the role of HcLec1 in innate immunity. We also established shell damage repair and pearl nucleus insertion models to examine the impact of HcLec1 on the biomineralization process in Hyriopsis cumingii Lea. In vitro calcium carbonate crystallization assays were conducted to explore the direct role of HcLec1 in calcium carbonate crystal formation. Results: The HcLec1 gene sequence is a full-length cDNA of 1552 bp, encoding 240 amino acids. HcLec1 comprises an N-terminal signal peptide and a carbohydrate-recognition domain (CRD), with QPD (Gln-Pro-Asp) and MND (Met-Asn-Asp) motifs for polysaccharide binding. Tissue expression analysis showed that HcLec1 is predominantly expressed in the gill tissue of Hyriopsis cumingii Lea under normal conditions, and its expression is significantly elevated in both gill and pearl sac tissues following nucleus insertion for pearl cultivation (P < 0.05). After immune stimulation with Aeromonas hydrophila and lipopolysaccharides (LPS), HcLec1 expression levels significantly increased in both cases (P < 0.01), indicating a role in bivalve innate immunity. RNA interference (RNAi)-mediated knockdown of HcLec1 led to a significant decrease in the expression levels of immune-related genes (WAP , α2m , and Lyso) and mineralization-related genes (CA , CHS , Nacrein , and Pif) (P < 0.05). In animal models for shell damage and nucleus insertion in pearl cultivation, HcLec1 showed a consistent expression pattern, with an initial significant decrease followed by a marked increase, peaking at day 14 (P < 0.05). This suggests a role for HcLec1 in pearl formation and shell repair. The recombinant HcLec1 protein demonstrated binding affinity to LPS and PGN, a robust ability to agglutinate Escherichia coli , Staphylococcus aureus , Aeromonas veronii , and Aeromonas hydrophila , and significantly inhibited bacterial growth (P < 0.05). Moreover, rHcLec1 promoted calcite crystal formation in saturated calcium carbonate solutions and altered crystal morphology. Discussion: The HcLec1 gene plays a pivotal role in both innate immunity and biomineralization in the triangle sail mussel. This study enhances our understanding of the functional diversity of C-type lectins and provides a foundation for future studies on shell repair and pearl growth. [ABSTRACT FROM AUTHOR]

The innate immune system plays a critical role in the rapid recognition and elimination of pathogens through pattern recognition receptors (PRRs). Among these PRRs are the C-type lectins (CTLs) langerin, mannan-binding lectin (MBL), and surfactant protein D (SP-D), which recognize carbohydrate patterns on pathogens. Each represents proteins from different compartments of the body and employs separate effector mechanisms. We have investigated their interaction with the Gram-positive opportunistic pathogen Staphylococcus aureus , a bacterium whose cell wall contains two key glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Using a langerin-expressing cell line and recombinant langerin, MBL, and SP-D, we demonstrated that langerin, MBL, and SP-D all recognize nonencapsulated S. aureus. However, the bacterium may produce CP that effectively shields S. aureus from recognition by all three CTLs. Experiments utilizing mutant S. aureus strains confirmed that WTA is a ligand for MBL, but that langerin likely interacts with an additional unknown ligand. A competition assay revealed that MBL and SP-D inhibit langerin's interaction with S. aureus , highlighting the intricate redundancy and cooperation within the innate immune system. This study highlights the dynamic interplay of langerin, MBL, and SP-D in recognizing specific surface structures on S. aureus and provides insight into how this pathogen evades innate immune recognition. [ABSTRACT FROM AUTHOR]

Galectins are widely distributed throughout the animal kingdom, from marine sponges to mammals. Galectins are a family of soluble lectins that specifically recognize β-galactoside-containing glycans and are categorized into three subgroups based on the number and function of their carbohydrate recognition domains (CRDs). The interaction of galectins with specific ligands mediates a wide range of biological activities, depending on the cell type, tissue context, expression levels of individual galectin, and receptor involvement. Galectins affect various immune cell processes through both intracellular and extracellular mechanisms and play roles in processes, such as apoptosis, angiogenesis, and fibrosis. Their importance has increased in recent years because they are recognized as biomarkers, therapeutic agents, and drug targets, with many other applications in conditions such as cardiovascular diseases and cancer. However, little is known about the involvement of galectins in liver diseases. Here, we review the functions of various galectins and evaluate their roles in liver diseases. [ABSTRACT FROM AUTHOR]

Introduction: Visualization of the intact microvascular network in skeletal muscle requires labeling the entire network in whole mount preparations where muscle fibre length can be set to near optimal but the tools to do this are not clear. Methods: We intravascularly injected CD-1 mice with different fluorescently labelled lectins (fluorescent isolectin GS-IB4 [ISO], wheat germ agglutinin [WGA], lycopersicon esculentum [LYCO]) or FITC-labelled gel. Soleus, extensor digitorum longus, diaphragm, gluteus maximus and cremaster muscles were excised, pinned at optimal sarcomere length and viewed using fluorescence microscopy. Results: WGA and LYCO were effective at labeling the entire vascular network with WGA labeling capillaries more brightly. ISO labelled the arteriolar vasculature and early segments of the capillaries but not the full length of the capillaries or the venular network. FITC-labelled gel was effective at labelling the microvascular network but not all small vessels were consistently labelled. The pattern of staining for each labelling method was similar across all muscle fibre-types tested. Conclusions: WGA was optimal for perfusion labeling and visualization of the intact microvascular network in whole mount skeletal muscle preparations and can be used in combination with ISO to distinguish the arteriolar and venous sides of the network. Introduction: Visualization of the intact microvascular network in skeletal muscle requires labeling the entire network in whole mount preparations where muscle fibre length can be set to near optimal but the tools to do this are not clear. Methods: We intravascularly injected CD-1 mice with different fluorescently labelled lectins (fluorescent isolectin GS-IB4 [ISO], wheat germ agglutinin [WGA], lycopersicon esculentum [LYCO]) or FITC-labelled gel. Soleus, extensor digitorum longus, diaphragm, gluteus maximus and cremaster muscles were excised, pinned at optimal sarcomere length and viewed using fluorescence microscopy. Results: WGA and LYCO were effective at labeling the entire vascular network with WGA labeling capillaries more brightly. ISO labelled the arteriolar vasculature and early segments of the capillaries but not the full length of the capillaries or the venular network. FITC-labelled gel was effective at labelling the microvascular network but not all small vessels were consistently labelled. The pattern of staining for each labelling method was similar across all muscle fibre-types tested. Conclusions: WGA was optimal for perfusion labeling and visualization of the intact microvascular network in whole mount skeletal muscle preparations and can be used in combination with ISO to distinguish the arteriolar and venous sides of the network. [ABSTRACT FROM AUTHOR]

Introduction: Ovarian cancer is a lethal disease with low survival rates for women diagnosed in advanced stages. Current cancer immunotherapies are not efficient in ovarian cancer, and there is therefore a significant need for novel treatment options. The β-galactoside-binding lectin, Galectin-3, is involved in different immune processes and has been associated with poor outcome in various cancer diagnoses. Here, we investigated how Galectin-3 affects the interaction between natural killer (NK) cells and neutrophils in the tumor microenvironment of ovarian cancer. Method: Ascites from the metastatic tumor microenvironment and cyst fluid from the primary tumor site were collected from patients with high-grade serous carcinoma (HGSC) together with peripheral blood samples. Galectin-3 concentration was measured in ascites, cyst fluid and serum or plasma. Neutrophils isolated from HGSC ascites and autologous blood were analyzed to evaluate priming status and production of reactive oxygen species. In vitro co-culture assays with NK cells, neutrophils and K562 target cells (cancer cell line) were conducted to evaluate NK cell viability, degranulation and cytotoxicity. Results: High levels of Galectin-3 were observed in cyst fluid and ascites from patients with HGSC. Neutrophils present in HGSC ascites showed signs of priming; however, the priming status varied greatly among the patient samples. Galectin-3 induced production of reactive oxygen species in ascites neutrophils, but only from a fraction of the patient samples, which is in line with the heterogenous priming status of the ascites neutrophils. In co-cultures with NK cells and K562 target cells, we observed that Galectin-3-induced production of reactive oxygen species in neutrophils resulted in decreased NK cell viability and lowered anti-tumor responses. Conclusion: Taken together, our results demonstrate high levels of Galectin-3 in the tumormicroenvironment of HGSC. High levels of Galectin-3 may induce production of reactiveoxygen species in ascites neutrophils in some patients. In turn, reactive oxygen species produced by neutrophils may modulate the NK cell anti-tumor immunity. Together, this study suggests further investigation to evaluate if a Galectin-3-targeting therapy may be used in ovarian cancer. [ABSTRACT FROM AUTHOR]

Background: Renal Cell Carcinoma is one of the most common malignancies worldwide. To date, multiple attempts had been made to accurately diagnose it and predict its behavior. One of the most intriguing biomarkers that have been assessed since 90s, is galectin-3. This study aimed to increase the prior knowledge of galectin-3 expression association with patient's clinicopathologic factors. Methods: In this single-center cross-sectional study, 71 patient samples from hospital archive were assessed for galectin-3 expression by immunohistochemistry assay. Pathologic slides were evaluated for histologic subtype, grade, stage, tumor size, presence of necrosis, and invasion of the renal vein. By adding cytoplasmic staining score to the color intensity score, a final score was recorded as galectin-3 positivity score. Results: 88 pathological slides of patients with confirmed RCC were screened and 71 were finally assessed. The mean age of the patients was 58.52 years (lowest 30 and highest 87). 67.6% were males and 32.4% were females. 68% of tumors were clear cell carcinoma, and only one oncocytoma was present. All 9 chromophobe cases showed a strong galectin-3 expression. Except for female gender (47.8% vs 18.8% in men; P=0.01), no statistically significant association was found between patient age, tumor grade, tumor size, tumor stage, and renal vein invasion with the level of galectin-3 expression. Conclusion: Considering the contradictory findings between this study and other similar studies, it can be concluded that the physiological role of galectins is very complex and the need for larger and more comprehensive evaluations is felt. [ABSTRACT FROM AUTHOR]

Lectins are non-covalent glycan-binding proteins found in all living organisms, binding specifically to carbohydrates through glycan-binding domains. Lectins have various biological functions, including cell signaling, molecular recognition, and innate immune responses, which play multiple roles in the physiological and developmental processes of organisms. Moreover, their diversity enables biotechnological exploration as biomarkers, biosensors, drug-delivery platforms, and lead molecules for anticancer, antidiabetic, and antimicrobial drugs. Lectins from Rhodophytes (red seaweed) have been extensively reported and characterized for their unique molecular structures, carbohydrate-binding specificities, and important biological activities. The increasing number of sequenced Rhodophyte genomes offers the opportunity to further study this rich source of lectins, potentially uncovering new ones with properties significantly different from their terrestrial plant counterparts, thus opening new biotechnological applications. We compiled literature data and conducted an in-depth analysis of the phycolectomes from all Rhodophyta genomes available in NCBI datasets. Using Hidden Markov Models capable of identifying lectin-type domains, we found at least six different types of lectin domains present in Rhodophytes, demonstrating their potential in identifying new lectins. This review integrates a computational analysis of the Rhodophyte phycolectome with existing information on red algae lectins and their biotechnological potential. [ABSTRACT FROM AUTHOR]

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Protein–carbohydrate interactions play a crucial role in mediating several biomolecular recognition events. We attempt to unravel its intricacies by understanding how carbohydrate‐binding proteins interpret the glycan code. We aim to decipher lectin‐mediated recognition in the endoplasmic reticulum (ER), which plays a crucial role in ER‐mediated quality control (ER‐QC). The ER‐QC functions in three phases—protein folding, transport, and degradation. Altered protein QC leads to ER‐related storage disorders. Cargo transport proteins—Ergic53 and Vip36—necessary for maintaining cellular homeostasis—are our primary focus. They recognize monoglucosylated/high mannose N‐glycans on the folded glycoproteins. This article reports on the first dynamic investigation of the ER cargo lectins in complex with the high mannose glycans using an advanced sampling technique—replica exchange molecular dynamics to decipher the inherent conformational heterogeneity and the binding mechanism. The study involves simulations for the proteins complexed with three high mannose glycans—Man8B, Man9, and mono‐glucosylated glycan. The recognition process is captured using MD simulations to achieve mechanistic insights and characterize the dynamics of glycans in their native and bound states via dihedral angle analysis. Results indicate that the flipped conformation of the glycans was crucial in differentiating their interaction with the proteins. Similar conformers of the glycans are preferred for Ergic53 and Vip36 in their glycan recognition events. Ergic53 preferred Man8B while it was Man9 for Vip36, in coherence with the previous experimental reports. These simulations provide a computational microscopic purview of the mechanism at both spatial and temporal scales. The results correlate with the published experimental data on the specificities of these lectins. [ABSTRACT FROM AUTHOR]

Galectins are β-galactosyl-binding lectins with key roles in early development, immune regulation, and infectious disease. Influenza A virus (IAV) infects the airway epithelia, and in severe cases may lead to bacterial superinfections and hypercytokinemia, and eventually, to acute respiratory distress syndrome (ARDS) through the breakdown of airway barriers. The detailed mechanisms involved, however, remain poorly understood. Our prior in vivo studies in a murine model system revealed that upon experimental IAV and pneumococcal primary and secondary challenges, respectively, galectin-1 and galectin-3 (Gal-3) are released into the airway and bind to the epithelium that has been desialylated by the viral neuraminidase, contributing to secondary bacterial infection and hypercytokinemia leading to the clinical decline and death of the animals. Here we report the results of in vitro studies that reveal the role of the extracellular Gal-3 in additional detrimental effects on the host by disrupting the integrity of the airway epithelial barrier. IAV infection of the human airway epithelia cell line A549 increased release of Gal-3 and its binding to the A549 desialylated cell surface, notably to the transmembrane signaling receptors CD147 and integrin-β1. Addition of recombinant Gal-3 to A549 monolayers resulted in enhanced expression and release of matrix metalloproteinases, leading to disruption of cell–cell tight junctions, and a significant increase in paracellular permeability. This study reveals a critical mechanism involving Gal-3 that may significantly contribute to the severity of IAV infections by promoting disruption of tight junctions and enhanced permeability of the airway epithelia, potentially leading to lung edema and ARDS. [ABSTRACT FROM AUTHOR]

Background: Over the past 40 years since the discovery of regenerating family proteins (Reg proteins), numerous studies have highlighted their biological functions in promoting cell proliferation and resisting cell apoptosis, particularly in the regeneration and repair of pancreatic islets and exocrine glands. Successively, short peptides derived from Reg3δ and Reg3α have been employed in clinical trials, showing favorable therapeutic effects in patients with type I and type II diabetes. However, continued reports have been limited, presumably attributed to the potential side effects. Methods: This review summarizes extensive research on Reg proteins over the past decade, combined with our own related studies, proposing that Reg proteins exhibit dimorphic effects. Results: The activity of Reg proteins is not as simplistic as previously perceived but shows auto-immunogenicity depending on different pathophysiological microenvironments. The immunogenicity of Reg proteins could recruit immune cells leading to an anti-tumor effect. Such functional diversity is correlated with their structural characteristics: the N-terminal region contributes to autoantigenicity, while the C-type lectin fragment near the C-terminal determines the trophic action. It should be noted that B-cell masking antigens might also reside within the C-type lectin domain. Conclusions: Reg proteins have dual functional roles under various physiological and pathological conditions. These theoretical foundations facilitate the subsequent development of diagnostic reagents and therapeutic drugs targeting Reg proteins. [ABSTRACT FROM AUTHOR]

In diabetes, tissue repair is impaired, increasing susceptibility to Staphylococcus aureus infections, a pathogen commonly found in wounds. The emergence of S. aureus strains that are highly resistant to antimicrobial agents highlights the urgent need for alternative therapeutic options. One promising candidate is Cramoll (Cratylia mollis seed lectin), known for its immunomodulatory, mitogenic, and healing properties. However, its efficacy in infected diabetic wounds remains unexplored. This study evaluated the effects of topical Cramoll treatment on diabetic wounds infected by S. aureus. Diabetic Swiss mice (induced by streptozotocin) were subjected to an 8-mm wound on the back and subsequently infected with a suspension of multidrug-resistant S. aureus. During the treatment period, the wounds were clinically evaluated for inflammation and the area of injury. After seven days, samples were collected from the wounds to quantify the bacterial load and histopathological and immunological analyses. Wounds infected by S. aureus exhibited more pronounced areas and severity indices, which were significantly reduced by Cramoll treatment (p < 0.05). Histopathological analysis revealed a reduction in inflammatory cells and an increase in revascularization with Cramoll treatment (p < 0.05). Cramoll also promoted greater collagen production compared to controls (p < 0.05). Furthermore, Cramoll treatment significantly reduced the S. aureus load in wounds (p < 0.0001), decreased TNF-α and IL-6 levels in infected wounds, and increased ERK pathway activation (p < 0.05). In conclusion, Cramoll lectin improves the healing of diabetic wounds, and these results contribute to the understanding of Cramoll healing mechanisms, reinforcing its potential as a healing agent in various clinical conditions. [ABSTRACT FROM AUTHOR]

Background: Inflammation markers in the blood may indicate a higher risk of unstable atherosclerosis. Selectins, a group of transmembrane glycoproteins, contribute to inflammation by helping certain blood cells bind to the endothelium. Methods: The study included 100 patients with stable early-onset coronary artery disease (CAD), 75 men (aged 50–54) and 25 women (aged 55–64). Tests performed included biochemical analysis, ultrasound, and Doppler imaging of arteries and peripheral vessels. A biochemical control group of 50 cases without CAD (74% men, average age 48 ± 3.20 years) was also studied. Results: Higher triglyceride levels were strongly linked to elevated plasma E-selectin levels. However, no significant relationship was found between plasma E-selectin levels and biochemical, clinical, radiographic, or echographic measures. Conclusion: Plasma E-selectin levels are not a reliable marker for detecting atherosclerotic plaques or related problems in individuals with stable, well-managed CAD. While E-selectin levels can be measured in clinical labs using immunoassays, they cannot replace standard cardiological and vascular imaging tests for diagnosing cardiac or vascular conditions. [ABSTRACT FROM AUTHOR]

Type 2 Diabetes Mellitus (T2DM) significantly contributes to cardiovascular disease (CVD) morbidity and mortality. This study examines the CVD risk among 276 T2DM patients using biomarker Galectin-3, along with clinical factors such as HbA1C levels, smoking, and alcohol consumption. The findings highlight the prevalence of moderate-to-high cardiovascular risk in this population, underscoring the need for targeted interventions. [ABSTRACT FROM AUTHOR]

Cancers utilize a simple glycan, Sialic Acid, to engage in metastatic processes via the Sialic acid (Sia) -Selectin pathway. Selectins recognize and bind to sialylated substrates, resulting in adhesion, migration, and extravasation, however, how deviations from the canonical form of Sia regulate binding to Selectin receptors (E, L, and P) on hemopoietic cells resulting in these metastatic processes, remained a gap in knowledge. De-O-acetylated Sias has been recently shown to be an integral substrate to the binding of sialic acid binding proteins. The two proteins responsible for regulating the acetyl functional group on Sia's C6 tail, are Sialic acid acetylesterase (SIAE) and Sialic acid O acetyltransferase (CASD1). To elucidate the contribution of functional group alterations on Sia, 9-O and 7,9-O-acetylation of Sia was modulated via the use of CRISRP-Cas9 gene editing and with Sialyl Glycan Recognition Probes, to produce either O-acetylated-Sia or de-O-acetylated- Sia, respectively. In vitro experiments revealed that increased cell surface expression of de-O-acetylated- Sia resulted in an increase in Selectin binding, enhanced cell proliferation, and increased migration capabilities both in lung and colon cancer. These results delineate for the first time the mechanistic contribution of de-O-acetylated-Sia to Selectin binding, reinforcing the importance of elucidating functional group alterations on Sia and their contribution. Without accurate identification of which functionalized form of Sia is being utilized to bind to sialic acid binding proteins, we cannot accurately produce glycan therapeutics with the correct specificity and reactivity, thus this work contributes an integral component in the development of promising therapeutic avenues, for example in the realm of enzyme antibody drug conjugates. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms which drive and modulate host-pathogen interactions are essential when designing effective therapeutic and diagnostic approaches aimed at controlling infectious diseases. Certain large and giant viruses have recently been discovered as components of the human virome, yet little is known about their interactions with the host immune system. We have dissected the role of viral N-linked glycans during the interaction between the glycoproteins from six chloroviruses (belonging to three chlorovirus classes: NC64A, SAG, and Osy viruses) and the representative carbohydrate-binding receptors of the innate immune system. Using solid-phase assays we have identified the binding of viral glycoproteins to different C-type lectins in a carbohydrate-dependent manner. These experiments verified the importance of D-rhamnose in modulating their binding to C-type lectins DC-SIGN and Langerin. In vitro assays further determined the ability of the chlorovirus glycoproteins to trigger secretion of cytokines Interleukins 6 and 10 (IL-6 and IL-10) in human monocyte-derived dendritic cells and mouse macrophages. Additionally, IgG from healthy human controls recognized certain chlorovirus glycoproteins, indicating the significance of human environmental viral exposures. Collectively, these results demonstrate the ability of the innate and adaptive immune systems to recognize chlorovirus glycoproteins, a process dependent on their specific N-glycan structures. This study explores how viral N-linked glycans play a role in the interaction between chloroviruses glycoproteins and key receptors of the innate immune system. [ABSTRACT FROM AUTHOR]

The cornea is densely innervated to maintain the integrity of the ocular surface, facilitating functions such as sensation and tear production. Following damage, alterations in the corneal microenvironment can profoundly affect its innervation, potentially impairing healing and sensory perception. One protein frequently upregulated at the ocular surface following tissue damage is galectin-3, but its contribution to corneal nerve regeneration remains unclear. Here, we sought to delineate the role of galectin-3 in regulating the expression of neurotrophic factors by different human cell types. Using a pathway-focused PCR array, we first evaluated the expression of neurotrophic factors in primary cultures of human corneal epithelial cells and fibroblasts. We found that these cell types contributed differently to the expression of these factors, with fibroblasts exhibiting higher levels of nerve growth factor , brain-derived neurotrophic factor , and GDNF compared to epithelial cells. Treatment with exogenous galectin-3 did not significantly affect epithelial cells; however, it did lead to increased synthesis and secretion of IL6, a cytokine known to influence neuronal survival and modulate inflammatory responses, by corneal fibroblasts. Using the human-derived SH-SY5Y cell line as a neuron-like cell model, we also found that galectin-3 stimulated the expression of FOS and LIF , two genes involved in neural differentiation and survival. In summary, these in vitro findings suggest that the presence of galectin-3 in the corneal environment may influence the neuronal response to injury. [ABSTRACT FROM AUTHOR]

Prolactin induced-protein (PIP) has been found to be rich in immunomodulatory epitopes, including N-acetyllactosamine (LacNAc) and N,N-diacetyllactosamine (LacdiNAc) residues, which may constitute ligands for galecin-3 (Gal-3). In the current study, we aimed to investigate the reactivity of galactose- and N-acetylgalactosamine-specific lectins with human seminal plasma PIP. Subsequently, we examined the direct interaction between seminal plasma PIP and galectin-3, and next analyzed whether there are any differences in the interaction associated with impaired semen parameters. The reactivity of terminal galactose-presenting glycans in seminal plasma PIP with Ricinus communis agglutinin I in the asthenozoospermic group was significantly higher compared to the normozoospermic fertile subjects. Investigating the reactivity of Wisteria floribunda lectin with PIP glycans, we found likewise significantly higher relative reactivity in the normozoospermic infertile as well as the oligoasthenozoopermic group compared to the control group. These results are related to the expression of LacdiNAc epitopes in the oligosaccharide chain of PIP. Finally, we observed that PIP reactivity with Wisteria floribunda lectin correlates positively with the interaction between galectin-3 and PIP in the seminal plasma. This can suggest that LacdiNAc residues are engaged in the interaction between PIP and galectin-3. [ABSTRACT FROM AUTHOR]

Recently, we reported that a recombinant Tepary bean (Phaseolus acutifolius) lectin (rTBL-1) induces apoptosis in colon cancer cell lines and that cytotoxicity was related to differential recognition of β1-6 branched N-glycans. Sequencing analysis and resolution of the rTBL-1 3D structure suggest that glycan specificity could be strongly influenced by two arginine residues, R103 and R130, located in the carbohydrate binding pocket. The aim of this work was to determine the contribution of these residues towards cytotoxic activity. Two rTBL-1 mutants were produced in Pichia pastoris, biochemically characterized, and cytotoxic effects were evaluated on human colorectal cancer cells (HT-29). Substitution of either of the arginine residues with glutamines resulted in significant reductions in cytotoxic activity, with losses of 1.5 and 3 times for R103 and R130, respectively. Docking analysis showed that the mutations decreased lectin affinity binding to some Epidermal Growth Factor Receptor (EGFR)-related N-glycans. Together, these findings confirm that both of the selected arginine residues (R103 and R130) play a key role in the recognition of tumor cell glycoconjugates by rTBL-1. [ABSTRACT FROM AUTHOR]

Purpose: Gestational diabetes mellitus (GDM) represents a significant metabolic disorder that affects pregnant women worldwide and has negative consequences for both the mother and her offspring. This research aims to investigate the relation between circulating levels of Galectin-3 and the incidence of GDM, and to evaluate its potential as a biomarker for monitoring and early detection of the disease. Methods: A thorough search of the literature has been performed using databases such as Scopus, Web of science, Embase, Cochrane Library and PubMed. The standardized mean difference (SMD) and corresponding confidence intervals (CIs) were used to compute the effect size from individual records and pooled using the Random-effect model. Results: Our meta-analysis synthesized data from 9 studies, encompassing 1,286 participants (533 GDM patients and 753 healthy pregnant controls). The findings demonstrated a considerable increase in Galectin-3 levels among individuals diagnosed with GDM as compared to the healthy control (SMD = 0.929; CI: 0.179–1.679; p = 0.015), with observed heterogeneity (I2 = 87%; p < 0.001). Subgroup analyses revealed the influence of factors such as age, BMI, study design, and sample type on Galectin-3 levels. A meta-regression analysis further identified trends indicating that levels of Galectin-3 are linked to gestational age, specific geographical areas, and sample size. Conclusion: Increased levels of Galectin-3 exhibit a significant association with GDM, indicating its prospective utility as a biomarker for early detection and risk assessment. Further research is warranted to elucidate its regulation and clinical implications in GDM management. [ABSTRACT FROM AUTHOR]

Quantitative transcriptomics offers a new way to obtain a detailed picture of freshly isolated cells. By direct isolation, the cells are unaffected by in vitro culture, and the isolation at cold temperatures maintains the cells relatively unaltered in phenotype by avoiding activation through receptor cross-linking or plastic adherence. Simultaneous analysis of several cell types provides the opportunity to obtain detailed pictures of transcriptomic differences between them. Here, we present such an analysis focusing on four human blood cell populations and compare those to isolated human skin mast cells. Pure CD19+ peripheral blood B cells, CD14+ monocytes, and CD4+ and CD8+ T cells were obtained by fluorescence-activated cell sorting, and KIT+ human connective tissue mast cells (MCs) were purified by MACS sorting from healthy skin. Detailed information concerning expression levels of the different granule proteases, protease inhibitors, Fc receptors, other receptors, transcription factors, cell signaling components, cytoskeletal proteins, and many other protein families relevant to the functions of these cells were obtained and comprehensively discussed. The MC granule proteases were found exclusively in the MC samples, and the T-cell granzymes in the T cells, of which several were present in both CD4+ and CD8+ T cells. High levels of CD4 were also observed in MCs and monocytes. We found a large variation between the different cell populations in the expression of Fc receptors, as well as for lipid mediators, proteoglycan synthesis enzymes, cytokines, cytokine receptors, and transcription factors. This detailed quantitative comparative analysis of more than 780 proteins of importance for the function of these populations can now serve as a good reference material for research into how these entities shape the role of these cells in immunity and tissue homeostasis. [ABSTRACT FROM AUTHOR]

The hemolymph of bivalve mollusks is rich in a large variety of lectins and other carbohydrate-binding proteins that function as agglutinins and provide effective protection against pathogens abundant in the aquatic environment. Screening of the agglutinating activity of hemolymph plasma was carried out for 19 bivalves species from eight orders (Adapedonta, Myida, Cardiida, Venerida, Arcida, Mytilida, Pectinida, and Ostreida) of two infraclasses (Heteroconchia and Pteriomorphia). Twelve types of erythrocytes, including six from marine mammals, were used in the hemagglutination test, which has made it possible to identify the agglutinating activity in all samples and conduct a valid study of their carbohydrate specificity using the most suitable erythrocytes in a hemagglutination inhibition assay. The characteristic carbohydrate specificity of bivalves' agglutinins to sialic and uronic acids, N-acetylated amines of glucose and galactose, mannan, and lactose has been shown. DL-Arabinose, D-xylose, D-glucurono-3,6-lactone, and α-methyl-D-glucopyranose have originally been found as specific molecular targets for proteins of some molluscan phylogenetic groups. These data foster isolation and identification of new carbohydrate-binding proteins capable of targeting specific glycosylation patterns valuable for biomedical and biotechnological applications. [ABSTRACT FROM AUTHOR]

Insect C‐type lectins (CTLs) play a vital role in recognizing and binding pathogen‐associated molecules, such as carbohydrate moieties, to initiate immune responses. Although the characteristics of Bombyx mori C‐type lectin 21 (BmCTL21), named BmLBP, have been reported, its molecular mechanisms, including expression patterns during immune responses and recognition and binding behaviors, remain poorly understood. Therefore, this study aims to explore the molecular characterization of BmCTL21 in innate immune responses. The findings reveal that BmCTL21 is upregulated in the fat body and subsequently transported to hemocytes, where it binds to the surface of Gram‐negative bacteria during microbial infection. Additionally, BmCTL21 RNAi treatment leads to a decline in BmCTL21 expression, which results in the suppression of phenoloxidase activity. Moreover, recombinant BmCTL21 was found to recognize carbohydrates, such as lipopolysaccharide, mannan and N‐acetyl‐ d‐glucosamine, in a Ca2+‐independent manner, and binds to the cell walls of Gram‐negative bacteria, Gram‐positive bacteria and fungi. In summary, this study offers new insights into the molecular mechanisms and functional behaviors of BmCTL21 in the innate immune response of silkworms. [ABSTRACT FROM AUTHOR]

Simple Summary: Smart, multifunctional nanoparticles (NPs) combine targeting strategies to overcome barriers and increase the efficacy of drug delivery. They can encapsulate anti-cancer therapeutic drugs, possess a targeting mechanism to locate malignant tissue effectively, and respond to stimuli releasing its cargo. The properties of these smart NPs ensure that the therapeutic drugs are not released before reaching target tissues and are only released at a specific rate to provide the most potent and sustained effect. Here, we designed and fabricated a functionalized folic acid (FA) conjugated poly(styrene-alt-maleic anhydride) (SMA) via biological linker 2,4-diaminobutyric acid (DABA) (FA–DABA–SMA) copolymer. The empty FA–DABA–SMA interactions with folic acid receptors (FRα) have been reported to disrupt intracellular signaling processes by dysregulating FRα, resulting in a secondary therapeutic mechanism of action. This process depends on the size and shape of the FA–DABA–SMA. In this study, we investigated if the FRα glycosylation profiles impact the FA–DABA–SMA binding and internalization of pancreatic PANC-1 cancer cells. Folate receptor alpha (FRα) is a glycosylphosphatidylinositol (GPI) membrane-anchored protein containing three N-glycosylated residues at the N47, N139, and N179 termini. These glycosylation sites have been reported to be crucial for the receptor's structural integrity and its ability to bind and internalize FA. Here, we investigated the role of FRα glycosylation in the binding and internalization efficacy of FA–DABA–SMA in pancreatic PANC-1 cancer cells. There is a strong association of the FA copolymer with FRα with a Pearson coefficient R-value of 0.7179. PANC-1 cancer cells were pretreated with maackia amurensis lectin II (MAL-2), sambucus Nigra lectin (SNA-1), peanut agglutinin (PNA), and wheat germ agglutinin lectin (WGA) at different doses followed by 20 kDa and 350 kDa FA–DABA–SMA loaded with coumarin 153 (C153). Increasing the dosage of MAL2, SNA-1, PNA, and WGA concomitantly and significantly increased the internalization of C153-loaded FA–DABA–SMA in the cells. The half maximal effective lectin concentrations (EC50) to induce cellular internalization into the cytoplasm of the lectins for MAL-2 were 35.88 µg/mL, 3.051 µg/mL for SNA-1, 7.883 µg/mL for PNA, and 0.898 µg/mL for WGA. Live cell imaging of the internalization of 20 kDa and 350 kDa FA copolymers indicated an aggregation of 350 kDa copolymer with FRα in the cytoplasm. In contrast, the 20 kDa FA copolymer remained in the membrane. The data indicate for the first time that the mobile positions of the glycosyl radical groups and the receptor tilt in generating steric hindrance impacted the individual FRα receptors in the binding and internalization of 350 kDa FA–DABA–SMA in cancer cells. [ABSTRACT FROM AUTHOR]