Receptors for carbohydrate ligands including heparin on the cell surface of Leishmania and other trypanosomatids

By employing neoglycoproteins (NGP) and glycosaminoglycans, the detection of endogenous glycoligand-binding sites has become possible. Monitoring specific binding of 11 of these sugar receptor-specific tools, 13 trypanosomatids of monogenetic genera Blastocrithidia, Crithidia, Herpetomonas , and Leptomonas and digenetic genera Endotrypanum , Leishmania , and Sauroleishmania were analysed by agglutination and fluorescence assays. NGP showed agglutination reactions only with the digenetic but not with the monogenetic species. Sensitive flow cytofluorimetric investigations revealed that the apparently different reactivity to NGP is due to a pronounced quantitative difference in expression of binding sites between mono- and digenetic flagellates. Moreover, flow cytofluorimetry was used to demonstrate the occurrence of receptor sites for heparin on the cell surfaces of all trypanosomatids. An indication for a correlation of the binding capacity for the NGP N-acetyl-β-d-glucosamine and heparin to differences in the pathogenicity of parasites was observed for Leishmania donovani as well as Leishmania enriettii . Infective populations of these species contained a significantly higher number of cells which had bound N-acetyl-β-d-glucosamine and heparin than noninfective (long-term in vitro -cultured) populations. The results of the present report additionally support the hypothesis that lectin–carbohydrate interactions with neutral sugar moieties and heparin or heparin-like molecules participate in the interactions between trypanosomatids and host (cells), and that the detected binding sites for carbohydrates and heparin may thus be referred to as potential virulence factors.     

Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell‐to‐cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl‐ and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA‐Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA‐Gal) binding activity was similar to that of BSA‐Fuc, but was weaker. In acrosome‐reacted spermatozoa treated with the Ca²⁺ ionophore A23187, BSA‐zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA‐Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml^?1 BSA‐Fuc, in vitro sperm–ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm–egg ZP interactions. At the same concentration, BSA‐Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm–ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.     

Determination of structural and functional overlap/divergence of five proto-type galectins by analysis of the growth-regulatory interaction with ganglioside GM1 in silico and in vitro on human neuroblastoma cells

The growth-regulatory interplay between ganglioside GM1 on human SK-N-MC neuroblastoma cells and an endogenous lectin provides a telling example for glycan (polysaccharide) functionality. Galectin-1 is the essential link between the sugar signal and the intracellular response. The emerging intrafamily complexity of galectins raises the question on defining extent of their structural and functional overlap/divergence. We address this problem for proto-type galectins in this system: ganglioside GM1 as ligand, neuroblastoma cells as target. Using the way human galectin-1 interacts with this complex natural ligand as template, we first defined equivalent positioning for distinct substitutions in the other tested proto-type galectins, e.g., Lys63 vs. Leu60/Gln72 in galectins-2 and -5. As predicted from our in silico work, the tested proto-type galectins have affinity for the pentasaccharide of ganglioside GM1. In contrast to solid-phase assays, cell surface presentation of the ganglioside did not support binding of galectin-5, revealing the first level of regulation. Next, a monomeric proto-type galectin (CG-14) can impair galectin-1-dependent negative growth control by competitively blocking access to the shared ligand without acting as effector. Thus, the quaternary structure of proto-type galectins is an efficient means to give rise to functional divergence. The identification of this second level of regulation is relevant for diagnostic monitoring. It might be exploited therapeutically by producing galectin variants tailored to interfere with galectin activities associated with the malignant phenotype. Moreover, the given strategy for comparative computational analysis of extended binding sites has implications for the rational design of galectin-type-specific ligands.     

Mannose 6‐phosphate‐modified bovine serum albumin nanoparticles for controlled and targeted delivery of sodium ferulate for treatment of hepatic fibrosis

Objectives The aim was to prepare neoglycoprotein‐based nanoparticles for targeted drug delivery to hepatic stellate cells, and to evaluate their characteristics in vitro and in vivo. Methods The neoglycoprotein of bovine serum albumin modified with mannose 6‐phosphate was synthesised from mannose, and used as wall material to nanoencapsulate the model natural antifibrotic substance sodium ferulate using a desolvation method. The morphology, drug loading capacity, release in vitro and biodistribution in vivo of the nanoparticles were studied. Selectivity of the nanoparticles for hepatic stellate cells was evaluated by immunohistochemical analysis of fibrotic rat liver sections. Key findings The spherical nanoparticles were negatively charged with zeta potential ranging from ?2.73 to ?35.85 mV, and sizes between 100 and 200 nm with a narrow size distribution. Drug entrapment efficiency of about 90% (w/w) and loading capacity of 20% (w/w) could be achieved. In vitro, the nanoparticles showed an initial rapid continuous release followed by a slower sustained release. After intravenous injection into mice, the nanoparticles showed a slower elimination rate and a much higher drug concentration in liver compared with the sodium ferrate solution, and less distribution to the kidneys and other tissues. Immunohistochemistry indicated that the neoglycoprotein‐based nano‐particles were taken up specifically by hepatic stellate cells. Conclusions The nanoparticles may be an efficient drug carrier targeting hepatic stellate cells.     

拟糖蛋白——肝炎治疗药物引导蛋白的合成

半乳糖基修饰的清蛋白可作为肝炎药物的引导蛋白,将药物浓集到肝实质细胞上。我们通过改进的方法制备2-亚氨基-2-甲氧基-乙基1-硫-半乳糖苷,并将此活性半孔糖衍生物连接到清蛋白上。所合成的糖蛋白经气相色谱测定,每分子蛋白上连接44个半乳糖基。     

Pregnancy following discontinuation of a calcium channel blocker in the male partner

The fertility potential of human sperm populations can be assessedby the presence of head-directed mannose ligand receptors (mannose-specificlectin) and the occurrence of spontaneous acrosome reactionsafter incubation under capacitating conditions in vitro. Wehave reported previously on the interaction between anti-hypertensivemedications and their effects on these parameters of male fertilitypotential. In this report we document the effects of cessationof calcium ion channel blocker medication on male fertility.Motile spermatozoa from a 30 year old infertile patient on acalcium ion channel blocker as anti-hypertensive treatment hadsubnormal expression of mannose-specific lectin and did notexhibit spontaneous acrosome reactions. Three months followingdiscontinuation of the medications, complete recovery of boththe expression of head-directed mannose ligand receptors andthe acrosome reaction was documented, though sperm motilityand morphology remained unchanged. The couple had 2 years ofinfertility and previously failed to conceive through sevencycles of Pergonal/intra-uterine insemination. Conception occurredon the second Pergonal/intra-uterine insemination cycle afterthe husband discontinued calcium ion channel blocker medication.Calcium ion channel blockers may adversely affect sperm fertilizingpotential. Discontinuation of such medications enhances thechances for conception.     

海洋真菌多糖YCP的拟糖蛋白制备及其免疫原性比较

目的：研究海洋真菌（PhomaherbarumYS4108）多糖YCP的免疫学性质。方法：将YCP与牛血清白蛋白（BSA）共价偶联，获得取代度（BSA／YCP）分别约为3．6和10的3种拟糖蛋白，经皮下分别接种ICR小鼠，测定免疫血清中多糖YCP的特异性抗体IgM和总IgG水平，以及半抗原和载体蛋白的特异性IgG亚型。结果：拟糖蛋白诱导小鼠产生强烈的抗YCP的IgM初次应答和IgG回忆应答。其中，抗多糖IgG亚型依次为IgG2a、IgG2b、IgG1和IgG3；而抗载体蛋白IgG亚型依次为IgG1、IgG2a、IgG2b和IgG3。结论：YCP诱导小鼠主要分泌IgG2a独特型抗体。     

Lectin-Mediated Drug Targeting: Selection of Valency, Sugar Type (Gal/Lac), and Spacer Length for Cluster Glycosides as Parameters to Distinguish Ligand Binding to C-Type Asialoglycoprotein Receptors and Galectins

Purpose. Common oligosaccharides of cellularglycoconjugates are ligands for more than one type of endogenous lectin.Overlapping specificities to -galactosides of C-type lectins andgalectins can reduce target selectivity of carbohydrate-ligand-dependentdrug targeting. The purpose of this study is to explore distinct features ofligand presentation and structure for design of cluster glycosides todistinguish between asialoglycoprotein-specific (C-type) lectins andgalectins. Methods. Extent of binding of labeled sugar receptors totwo types of matrix-immobilized (neo)glycoproteins and to cells wasevaluated in the absence and presence of competitive inhibitors. This panelcomprised synthetic mono-, bi-, and trivalent glycosides with two spacerlengths and galactose or lactose as ligand part. Results. In contrast to C-type lectins of hepatocytes andmacrophages, bi- and trivalent glycosides do not yield a notable glycosidecluster effect for galectins-1 and -3. Also, theseCa²⁺-independent galactoside-binding proteins prefer to homein on lactose-bearing glycosides relative to galactose as ligand, whilespacer length requirements were rather similar. Conclusions. Trivalent cluster glycosides with Gal/GalNAcas ligand markedly distinguish between C-type lectins and galectins.Undesired side reactivities to galectins for C-type lectin drug deliverywill thus be minimal.