ResearchPad - lectins https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[3D-Reconstruction of the human conventional outflow system by ribbon scanning confocal microscopy]]> https://www.researchpad.co/article/elastic_article_15723 The risk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma.MethodsAfter perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data.ResultsRSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemm’s canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized.ConclusionIn this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma. ]]> <![CDATA[R pyocin tail fiber structure reveals a receptor-binding domain with a lectin fold]]> https://www.researchpad.co/article/5c633948d5eed0c484ae63f5

R pyocins are ɸCTX-like myophage tailocins of Pseudomonas sp. Adsorption of R pyocins to target strains occurs by the interaction of tail fiber proteins with core lipopolysaccharide (LPS). Here, we demonstrate that N-terminally truncated R pyocin tail fibers corresponding to a region of variation between R-subtypes are sufficient to bind target strains according to R-subtype. We also report the crystal structures of these tail fiber proteins and show that they form an elongated helical trimer composed of three domains arranged linearly from N- to C-terminus: a baseplate proximal head, medial shaft, and distal foot. The head and shaft domains contain novel structural motifs. The foot domain, however, is composed of a conserved jellyroll fold and shares high structural similarity to the tail fiber of myophage AP22, podophage tailspike C-terminal domains (LKA-1 and ɸ297), and several eukaryotic adhesins (discoidin I/II, agglutinin, and octocoral lectin). Many of these proteins bind polysaccharides by means of their distal loop network, a series of highly variable loops at one end of the conserved jellyroll fold backbone. Our structures reveal that the majority of R-subtype specific polymorphisms cluster in patches covering a cleft formed at the oligomeric interface of the head domain and in a large patch covering much of the foot domain, including the distal loop network. Based on the structural variation in distal loops within the foot region, we propose that the foot is the primary sugar-binding domain of R pyocins and R-subtype specific structural differences in the foot domain distal loop network are responsible for binding target strains in an R-subtype dependent manner.

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<![CDATA[Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells]]> https://www.researchpad.co/article/5c4b7f2dd5eed0c484840b72

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

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<![CDATA[COLEC10 is mutated in 3MC patients and regulates early craniofacial development]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdce23

3MC syndrome is an autosomal recessive heterogeneous disorder with features linked to developmental abnormalities. The main features include facial dysmorphism, craniosynostosis and cleft lip/palate; skeletal structures derived from cranial neural crest cells (cNCC). We previously reported that lectin complement pathway genes COLEC11 and MASP1/3 are mutated in 3MC syndrome patients. Here we define a new gene, COLEC10, also mutated in 3MC families and present novel mutations in COLEC11 and MASP1/3 genes in a further five families. The protein products of COLEC11 and COLEC10, CL-K1 and CL-L1 respectively, form heteromeric complexes. We show COLEC10 is expressed in the base membrane of the palate during murine embryo development. We demonstrate how mutations in COLEC10 (c.25C>T; p.Arg9Ter, c.226delA; p.Gly77Glufs*66 and c.528C>G p.Cys176Trp) impair the expression and/or secretion of CL-L1 highlighting their pathogenicity. Together, these findings provide further evidence linking the lectin complement pathway and complement factors COLEC11 and COLEC10 to morphogenesis of craniofacial structures and 3MC etiology.

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<![CDATA[Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array]]> https://www.researchpad.co/article/5989db41ab0ee8fa60bd6ef4

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.

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<![CDATA[Lectin-Like Molecules of Lactobacillus rhamnosus GG Inhibit Pathogenic Escherichia coli and Salmonella Biofilm Formation]]> https://www.researchpad.co/article/5989da50ab0ee8fa60b8dc16

Objectives

Increased antibiotic resistance has catalyzed the research on new antibacterial molecules and alternative strategies, such as the application of beneficial bacteria. Since lectin molecules have unique sugar-recognizing capacities, and pathogens are often decorated with sugars that affect their survival and infectivity, we explored whether lectins from the probiotic strain Lactobacillus rhamnosus GG have antipathogenic properties.

Methods

The genome sequence of L. rhamnosus GG was screened for the presence of lectin-like proteins. Two genes, LGG_RS02780 and LGG_RS02750, encoding for polypeptides with an N-terminal conserved L-type lectin domain were detected and designated Llp1 (lectin-like protein 1) and Llp2. The capacity of Llp1 and Llp2 to inhibit biofilm formation of various pathogens was investigated. Sugar specificity was determined by Sepharose beads assays and glycan array screening.

Results

The isolated lectin domains of Llp1 and Llp2 possess pronounced inhibitory activity against biofilm formation by various pathogens, including clinical Salmonella species and uropathogenic E. coli, with Llp2 being more active than Llp1. In addition, sugar binding assays with Llp1 and Llp2 indicate specificity for complex glycans. Both proteins are also involved in the adhesion capacity of L. rhamnosus GG to gastrointestinal and vaginal epithelial cells.

Conclusions

Lectins isolated from or expressed by beneficial lactobacilli could be considered promising bio-active ingredients for improved prophylaxis of urogenital and gastrointestinal infections.

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<![CDATA[Hepatitis C Virus Resistance to Carbohydrate-Binding Agents]]> https://www.researchpad.co/article/5989da57ab0ee8fa60b8f1ab

Carbohydrate binding agents (CBAs), including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA), Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms.

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<![CDATA[Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography]]> https://www.researchpad.co/article/5989da30ab0ee8fa60b844ca

Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.

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<![CDATA[Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1]]> https://www.researchpad.co/article/5989dae0ab0ee8fa60bbb913

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.

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<![CDATA[GWAS for serum galactose-deficient IgA1 implicates critical genes of the O-glycosylation pathway]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdd092

Aberrant O-glycosylation of serum immunoglobulin A1 (IgA1) represents a heritable pathogenic defect in IgA nephropathy, the most common form of glomerulonephritis worldwide, but specific genetic factors involved in its determination are not known. We performed a quantitative GWAS for serum levels of galactose-deficient IgA1 (Gd-IgA1) in 2,633 subjects of European and East Asian ancestry and discovered two genome-wide significant loci, in C1GALT1 (rs13226913, P = 3.2 x 10−11) and C1GALT1C1 (rs5910940, P = 2.7 x 10−8). These genes encode molecular partners essential for enzymatic O-glycosylation of IgA1. We demonstrated that these two loci explain approximately 7% of variability in circulating Gd-IgA1 in Europeans, but only 2% in East Asians. Notably, the Gd-IgA1-increasing allele of rs13226913 is common in Europeans, but rare in East Asians. Moreover, rs13226913 represents a strong cis-eQTL for C1GALT1 that encodes the key enzyme responsible for the transfer of galactose to O-linked glycans on IgA1. By in vitro siRNA knock-down studies, we confirmed that mRNA levels of both C1GALT1 and C1GALT1C1 determine the rate of secretion of Gd-IgA1 in IgA1-producing cells. Our findings provide novel insights into the genetic regulation of O-glycosylation and are relevant not only to IgA nephropathy, but also to other complex traits associated with O-glycosylation defects, including inflammatory bowel disease, hematologic disease, and cancer.

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<![CDATA[Thermal, Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation]]> https://www.researchpad.co/article/5989db47ab0ee8fa60bd8bed

Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol−1 and 14.90 Kcal mol−1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp) is 3.42 Kcal mol−1 K−1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

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<![CDATA[Modulation of functional characteristics of resident and thioglycollate-elicited peritoneal murine macrophages by a recombinant banana lectin]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbb9e

We demonstrated that a recombinant banana lectin (rBanLec), which structural characteristics and physiological impacts highly resemble those reported for its natural counterparts, binds murine peritoneal macrophages and specifically modulates their functional characteristics. By using rBanLec in concentrations ranging from 1 μg to 10 μg to stimulate resident (RMs) and thioglycollate-elicited (TGMs) peritoneal macrophages from BALB/c and C57BL/6 mice, we have shown that effects of rBanLec stimulation depend on its concentration but also on the functional status of macrophages and their genetic background. rBanLec, in a positive dose-dependent manner, promotes the proliferation of TGMs from both BALB/c and C57BL/6 mice, while its mitogenic influence on RMs is significantly lower (BALB/c mice) or not detectable (C57BL/6 mice). In all peritoneal macrophages, irrespective of their type and genetic background, rBanLec, in a positive dose dependent manner, enhances the secretion of IL-10. rBanLec stimulation of RMs from both BALB/c and C57BL/6 resulted in a positive dose-dependent promotion of proinflammatory phenotype (enhancement of NO production and IL-12 and TNFα secretion, reduction of arginase activity). Positive dose-dependent skewing toward proinflammatory phenotype was also observed in TGMs from C57BL/6 mice. However, the enhancement of rBanLec stimulation promotes skewing of TGMs from BALB/c mice towards anti-inflammatory profile (reduction of NO production and IL-12 secretion, enhancement of arginase activity and TGFβ and IL-4 secretion). Moreover, we established that rBanLec binds oligosaccharide structures of TLR2 and CD14 and that blocking of signaling via these receptors significantly impairs the production of TNFα and NO in BALB/c macrophages. Since the outcome of rBanLec stimulation depends on rBanLec concentration as well as on the functional characteristics of its target cells and their genetic background, further studies are needed to investigate its effects under physiological and specific pathological conditions.

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<![CDATA[Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili]]> https://www.researchpad.co/article/5989d9f6ab0ee8fa60b7030a

Mapping of the microbial molecules underlying microbiota-host interactions is key to understand how microbiota preserve mucosal homeostasis. A pivotal family of such bacterial molecules are pili. Pili are proteinaceous cell wall appendages with a well-documented role in adhesion, whilst their role in immune interaction with the host is less established. Gram-positive pili are often posttranslationally modified by sortase-specific cleavage reactions and the formation of intramolecular peptide bonds. Here we report glycosylation as a new level of posttranslational modification of sortase-dependent pili of a beneficial microbiota species and its role in immune modulation. We focused on the SpaCBA pili of the model probiotic and beneficial human gut microbiota isolate Lactobacillus rhamnosus GG. A unique combination of molecular techniques, nanoscale mechanical and immunological approaches led to the identification of mannose and fucose residues on the SpaCBA pili. These glycans on the pili are recognized by human dendritic cells via the C-type lectin receptor DC-SIGN, a key carbohydrate-dependent immune tailoring pattern recognition receptor. This specific lectin-sugar interaction is moreover of functional importance and modulated the cytokine response of dendritic cells. This provides insight into the direct role bacterial glycoproteins can play in the immunomodulation of the host. Modification of the complex heterotrimeric pili of a model probiotic and microbiota isolate with mannose and fucose is of importance for the functional interaction with the host immune lectin receptor DC-SIGN on human dendritic cells. Our findings shed light on the yet underappreciated role of glycoconjugates in bacteria-host interactions.

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<![CDATA[Gelation of Soy Milk with Hagfish Exudate Creates a Flocculated and Fibrous Emulsion- and Particle Gel]]> https://www.researchpad.co/article/5989d9fcab0ee8fa60b7257f

Hagfish slime is an ultra dilute, elastic and cohesive hydrogel that deploys within milliseconds in cold seawater from a glandularly secreted exudate. The slime is made of long keratin-like fibers and mucin-like glycoproteins that span a network which entraps water and acts as a defense mechanism against predators. Unlike other hydrogels, the slime only confines water physically and is very susceptible to mechanical stress, which makes it unsuitable for many processing operations and potential applications. Despite its huge potential, little work has been done to improve and functionalize the properties of this hydrogel. To address this shortcoming, hagfish exudate was mixed with a soy protein isolate suspension (4% w/v) and with a soy emulsion (commercial soy milk) to form a more stable structure and combine the functionalities of a suspension and emulsion with those of the hydrogel. Hagfish exudate interacted strongly with the soy systems, showing a markedly increased viscoelasticity and water retention. Hagfish mucin was found to induce a depletion and bridging mechanism, which caused the emulsion and suspension to flocculate, making “soy slime”, a cohesive and cold-set emulsion- and particle gel. The flocculation network increases viscoelasticity and substantially contributes to liquid retention by entrapping liquid in the additional confinements between aggregated particles and protein fibers. Because the mucin-induced flocculation resembles the salt- or acid-induced flocculation in tofu curd production, the soy slime was cooked for comparison. The cooked soy slime was similar to conventional cooked tofu, but possessed a long-range cohesiveness from the fibers. The fibrous, cold-set, and curd-like structure of the soy slime represents a novel way for a cold coagulation and fiber incorporation into a suspension or emulsion. This mechanism could be used to efficiently gel functionalized emulsions or produce novel tofu-like structured food products.

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<![CDATA[A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen-Associated Molecular Patterns of Bacteria through Rhamnose]]> https://www.researchpad.co/article/5989da46ab0ee8fa60b8bc29

Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.

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<![CDATA[Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells]]> https://www.researchpad.co/article/5989da80ab0ee8fa60b9a32c

Complementary treatment possibilities for the therapy of cancer are increasing in demand due to the severe side effects of the standard cytostatics used in the first-line therapy. A common approach as a complementary treatment is the use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic activity of these extracts is attributed to Mistletoe lectins which are Ribosome-inactivating proteins type II. Besides these main constituents the extract of Viscum album L. comprises also a mixture of lipophilic ingredients like triterpene acids of the oleanane, lupane and ursane type. However, these constituents are not contained in commercially available aqueous extracts due to their high lipophilicity and insolubility in aqueous extraction media. To understand the impact of the extract ingredients in cancer therapy, the intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells was investigated in relation to the mistletoe triterpene acids, mainly oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized using cyclodextrins (“TT” extract). Afterwards, the uptake of either single compounds (isolated ML and the aqueous “viscum” extract) or in combination with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30, 60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies the A-chain of the hololectin. It could be shown that the intracellular uptake after 120 minutes amounted to 20% in all cell lines after incubation with viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and Ewing TC-71-cells was independent of the addition of TT extract. Interestingly, the uptake of ML by 143B-cells could only be measured after addition of triterpenes pointing to resistance to mistletoe lectin.

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<![CDATA[Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc8e0

Carbohydrate–protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6 pN and 7.1 pN were determined for the MUC1(Tn)—MGL and MUC1(STn)—MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27 and 37 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.

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<![CDATA[Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor]]> https://www.researchpad.co/article/5989da78ab0ee8fa60b9778b

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.

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<![CDATA[Regional early and progressive loss of brain pericytes but not vascular smooth muscle cells in adult mice with disrupted platelet-derived growth factor receptor-β signaling]]> https://www.researchpad.co/article/5989db59ab0ee8fa60bdf0f0

Pericytes regulate key neurovascular functions of the brain. Studies in pericyte-deficient transgenic mice with aberrant signaling between endothelial-derived platelet-derived growth factor BB (PDGF-BB) and platelet-derived growth factor receptor β (PDGFRβ) in pericytes have contributed to better understanding of the role of pericytes in the brain. Here, we studied PdgfrβF7/F7 mice, which carry seven point mutations that disrupt PDGFRβ signaling causing loss of pericytes and vascular smooth muscle cells (VSMCs) in the developing brain. We asked whether these mice have a stable or progressive vascular phenotype after birth, and whether both pericyte and VSMCs populations are affected in the adult brain. We found an early and progressive region-dependent loss of brain pericytes, microvascular reductions and blood-brain barrier (BBB) breakdown, which were more pronounced in the cortex, hippocampus and striatum than in the thalamus, whereas VSMCs population remained unaffected at the time when pericyte loss was already established. For example, compared to age-matched controls, PdgfrβF7/F7 mice between 4–6 and 36–48 weeks of age developed a region-dependent loss in pericyte coverage (22–46, 24–44 and 4–31%) and cell numbers (36–49, 34–64 and 11–36%), reduction in capillary length (20–39, 13–46 and 1–30%), and an increase in extravascular fibrinogen-derived deposits (3.4–5.2, 2.8–4.1 and 0–3.6-fold) demonstrating BBB breakdown in the cortex, hippocampus and thalamus, respectively. Capillary reductions and BBB breakdown correlated with loss of pericyte coverage. Our data suggest that PdgfrβF7/F7 mice develop an aggressive and rapid vascular phenotype without appreciable early involvement of VSMCs, therefore providing a valuable model to study regional effects of pericyte loss on brain vascular and neuronal functions. This model could be a useful tool for future studies directed at understanding the role of pericytes in the pathogenesis of neurological disorders associated with pericyte loss such as vascular dementia, Alzheimer’s disease, amyotrophic lateral sclerosis, stroke and human immunodeficiency virus-associated neurocognitive disorder.

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<![CDATA[Carbohydrate-Binding Non-Peptidic Pradimicins for the Treatment of Acute Sleeping Sickness in Murine Models]]> https://www.researchpad.co/article/5989dabeab0ee8fa60baf9e0

Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT.

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