Galactosyl Moiety Research Articles

C.376A>G, (p.Ser126Gly) Alpha-Galactosidase A mutation induces ER stress, unfolded protein response and reduced enzyme trafficking to lysosome: Possible relevance in the pathogenesis of late-onset forms of Fabry Disease

Fabry Disease (FD) (OMIM 301500) is a metabolic X-linked inherited lysosomal storage disorder that results from the deficient activity of Alpha-Galactosidase A (Alpha-Gal), a lysosomal hydrolase that cleaves neutral glycosphingolipids with terminal N-linked galactosyl moieties, mainly globotriaosylceramides (Gb3). The enzyme, encoded by a 12-kb gene mapping on the long arm (Xq22.1 region) of the X chromosome, is constituted by a glycosylated subunit of approximately 55 kD, synthesized as an inactive precursor that undergoes maturation in endoplasmic reticulum (ER) and Golgi apparatus before being delivered to the lysosome to form a functional dimer.The gene is comprised of seven exons and, so far, >1000 different mutations have been described as associated to FD (www.dbfgp.org/dbFgp/fabry/FabryGP.htm).Clinical phenotypes are divided in two main classes, classic or non-classic, based on clinical and biochemical findings. Non-classic FD, usually recognized as late-onset forms with oligosymptomatic phenotype, presents with symptoms restricted solely to cardiocytes, kidneys or brain associated to missense misfolding mutations. In the group of the non-classic FD, special attention should be given to patients carrying the c.376A > G (p.Ser126Gly) mutation. The lack of clear experimental evidences on its pathogenetic role, despite the clinical pictures of the patients with severe ischaemic lesions, renal involvement and acroparesthesias, led many authors to classify this mutation as inconsistent, non-pathogenetic, and consequently not eligible to the current pharmacological treatments for FD. To shed light on the cellular processes affected by this mutation and to assess if the biochemical pathways involved with, could really have a significant pathogenetic impact, we studied the mutation in silico and in COS-7 and HEK 293 cell models. We found p.Ser126Gly, even retaining both high degree of synthesis and residual activity, is mostly stacked into the ER inducing unfolded protein response (UPR) with reduced trafficking to the lysosome. These data strongly suggest that p.Ser126Gly could trigger a pathogenetic mechanism different from the classic and well assessed increased turnover with loss of biological activity described for other missense mutations. This mechanism seems mainly related to a negative gain of function, with ER retention and UPR activation and could lead, via inflammation and/or apoptosis, to irreversible cell damage.

First-in-Class Dual Targeting Compounds for the Management of Seizures in Glucose Transporter Type 1 Deficiency Syndrome.

The genetic disorder glucose transporter type 1 deficiency syndrome (GLUT1-DS) heavily affects the main intake of energy in tissues and determines the most relevant outcomes at the central nervous system (CNS) district, which is highly dependent on glucose. Herein, we report the design and development of a set of compounds bearing the glucosyl and galactosyl moieties. We assessed their ability to enhance the GLUT1 mediated glucose intake in non-small-cell lung cancer (NSCLC) cells and to inhibit the carbonic anhydrase (CA; EC 4.2.1.1) isoforms implicated in the physiopathology of uncontrolled seizures associated to epilepsy (i.e., I, II, IV, VA, VB, and XII). The binding mode of 8 in adduct with hCA II was determined by X-ray crystallography. Among the selected derivatives, compound 4b proved effective in suppressing the occurrence of uncontrolled seizures on the in vivo induced maximal electroshock (MES) model and thus gives sustainment of an unprecedently reported pharmacological approach for the management of GLUT1-DS associated diseases.

Stereoselective Synthesis of New 4-Aryl-5-indolyl-1,2,4-triazole S- and N-β-Galactosides: Characterizations, X-ray Crystal Structure and Hirshfeld Surface Analysis

5-(1H-Indol-2-yl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione 1a and 4-(4-chlorophenyl)-5-(1H-indol-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 1b were galactosylated in the presence of NaHCO3 in ethanol to produce S-galactosides 3,4, whereas, in the presence of K2CO3 in acetone they produced a mixture of S- and N-galactosides 3-6 with a higher yield of S-galactosides over the respective N-galactosides. Improvement in the yields of N-galactosides was produced by thermal migration of the galactosyl moiety from sulfur to nitrogen using fusion. β-Stereoselectivity of galactosylation was determined using the coupling constant value 3J1,2, which exceeded 9.0 Hz in all prepared galactosides. The precursors 1a and 1b alkylated with 3-bromopropan-1-ol 7 in K2CO3 and acetone produced the S-alkylated products 8 and 9, respectively. Structural determinations of new compounds 5 and 9 are presented. The phenyl and indole moieties were found to be twisted from the triazole ring mean in both compounds. For compound 5, the twist angles were 66.24° and 18.86°, respectively, while the corresponding values for 9 were in the ranges of 73.15–77.29° and 13.96–20.70°, respectively. Hence, the crystal system of 9 is triclinic while the space group is P-1. Detailed analysis of the intermolecular interactions in the crystal structure of 5 is presented using Hirshfeld calculations. The O…H, N…H, C…H, and S…H contacts appeared as red spots in the dnorm Hirshfeld surface indicating short distance intermolecular interactions. Their percentages were estimated based on the decomposition of the fingerprint plot to be 25.6, 2.4, 14.0, and 6.3%, respectively.

Galectin-1 mediates interactions between polymorphonuclear leukocytes and vascular endothelial cells, and promotes their extravasation during lipopolysaccharide-induced acute lung injury.

The lung airway epithelial surface is heavily covered with sialic acids as the terminal carbohydrate on most cell surface glycoconjugates and can be removed by microbial neuraminidases or endogenous sialidases. By desialylating the lung epithelial surface, neuraminidase acts as an important virulence factor in many mucosal pathogens, such as influenza and S. pneumoniae. Desialylation exposes the subterminal galactosyl moieties - the binding glycotopes for galectins, a family of carbohydrate-recognition proteins playing important roles in various aspects of immune responses. Galectin-1 and galectin-3 have been extensively studied in their roles related to host immune responses, but some questions about their role(s) in leukocyte recruitment during lung bacterial infection remain unanswered. In this study, we found that both galectin-1 and galectin-3 bind to polymorphonuclear leukocytes (PMNs) and enhance the interaction of endothelial intercellular adhesion molecule-1 (ICAM-1) with PMNs, which is further increased by PMN desialylation. In addition, we observed that in vitro galectin-1 mediates the binding of PMNs, particularly desialylated PMNs, onto the endothelial cells. Finally, in a murine model for LPS-mediated acute lung injury, we observed that galectin-1 modulates PMN infiltration to the lung without altering the expression of chemoattractant cytokines. We conclude that galectins, particularly galectin-1, may function as adhesion molecules that mediate PMN-endothelial cell interactions, and modulate PMN infiltration during acute lung injury.

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

Arsenic-induced galactinol synthase1 gene, AtGolS1, provides arsenic stress tolerance in Arabidopsis thaliana

Combating stress conditions and maintaining cellular homeostasis are integral to plant growth and development. To maintain cellular integrity, defence mechanisms against abiotic stresses often work by accumulating molecules, including antioxidants, secondary metabolites, and soluble sugars. The Raffinose Family of Oligosaccharide (RFO) biosynthesis is an extension of sucrose metabolism involving galactinol synthase (GolS). GolS transfers galactosyl moieties to myo-inositol to generate Galactinol, followed by the formation of Raffinose and Stachyose. GolS, being the rate-limiting enzyme, is often targeted to understand this pathway's role under different stress conditions; however, till now, the involvement of GolS1 in heavy metal stress remains unknown. This study demonstrates an increase in AtGolS1 expression and Galactinol accumulation in response to arsenic (As) stress in Arabidopsis thaliana. Analysis of transgenic lines over-expressing AtGolS1 revealed an improved growth under As stress. Contrasting sensitivity by Arabidopsis mutant under As further validated the role of AtGolS1 in combating As stress. Response of transgenic lines expressing a reporter gene, GUS, under the AtGolS1 promoter suggests responsiveness of GolS1 to As stress. This study implies that AtGolS1 is inducible by As stress at the transcriptional and metabolic levels, possibly to mitigate excessive ROS production and provide tolerance under As stress in plants.

Asn215Ser, Ala143Thr, and Arg112Cys variants in α-galactosidase A protein confer stability loss in Fabry’s disease

Alpha galactosidase A (α-GalA) gene contains nine exons localized at the q-arm of the X chromosome. Generally, an α-GalA enzyme is involved in the removal of galactosyl moieties from the glycoproteins and glycolipids. Dysregulation results in the accumulation of glycoproteins as well as glycolipids in various organs leading to Fabry disease (FD). In this study, we examine the impact of Asn215Ser, Ala143Thr and Arg112Cys variants on the α-GalA protein structure contributing to functional dynamic changes in FD. The seven computational pathogenicity prediction methods were used to predict the effects of these variants on the α-GalA protein. The three-dimensional structure of α-GalA variants was modeled with the Swiss Model and Robetta server and validated using a variety of tools. Then, molecular dynamics (MD) simulation was performed to understand the stability and dynamic behavior of the wild-type and variants structures. Most of our analyzed pathogenicity prediction tools showed that Asn215Ser, Ala143Thr and Arg112Cys variants cause a deleterious effect on the α-GalA protein. Further, MD trajectory analysis showed the destabilizing effect of variants on α-GalA structure based on the root mean square deviation, root mean square fluctuation, solvent accessible surface area, the radius of gyration, hydrogen bond, cluster analysis and PCA analysis. This concludes that the presence of these variants could potentially affect the protein functional process of galactosyl moieties removal which might lead to Fabry disease. Communicated by Ramaswamy H. Sarma

Novel Galactosyl Moiety-Conjugated Furylchalcones Synthesized Facilely Display Significant Regulatory Effect on Plant Growth.

The expansion of weed infestation has increased the demand on new herbicides. A series of novel galactosyl moiety-conjugated furylchalcones was facilely synthesized in which the furyl group (A ring) was combined with the substituted benzene group (B ring), and a galactosyl moiety was introduced. All these galactosyl furylchalcones were predicted to be phloem-mobile. Most of the galactosyl furylchalcones significantly promoted early seedling growth of sorghum and barnyardgrass under dark conditions, but all of them revealed considerable anti-growth ability on illuminated pot plants; especially, 1-(3'-(4″-O-β-d-galactopyranosyl)furyl)-3-(4″-nitrophenyl)-2-en-1-one (B11) had a better herbicidal activity against rapeseed and Chinese amaranth than haloxyfop-R-methyl. The median efficient concentrations (EC50) of compound B11 against cucumber and wheat were 9.55 and 26.97 mg/L, respectively, also showing a stronger suppressing capacity than 2,4-D. Molecular docking with phosphoenolpyruvate carboxylase protein showed a stable binding conformation in which the galactosyl group interacted with LYS363 and GLU369, the furan ring and carbonyl bound with ARG184, and the crosslink of the nitro group with GLU240 formed a salt bridge. The results demonstrate that galactosyl furylchalcones possess the great potential as new herbicides for weed management, and further evaluations on more weeds are required for practical application.

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

Mannosyl, glucosyl or galactosyl liposomes to improve resveratrol efficacy against Methicillin Resistant Staphylococcus aureus biofilm

Novel cationic glycoliposomes, composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and glycoamphiphiles featuring a galactosyl, mannosyl or glucosyl moiety have been investigated for the targeted delivery of trans-resveratrol (RSV), a Quorum Sensing Inhibitor (QSI), to Methicillin Resistant Staphylococcus Aureus (MRSA) biofilms.All the glycosylated formulations show a 10–20 % reduction of their hydrodynamic size and a high positive increase in ζ-potential (20÷27mV), with respect to the almost neutral DOPC/Chol liposomes (-3.7 mV). RSV is entrapped in liposomes with high Entrapment Efficiency (EE%), the formulations containing glycosylated amphiphiles showing higher values of EE% (79–90 %) than those containing DOPC/CHOL (65 %). In all the liposomal formulations, the inclusion of RSV causes a decrease in ζ-potential, which is particularly evident in the negative value of DOPC/Chol liposomes (-14.8 mV). This is probably due to the ionization of a small percentage of RSV molecules that point towards the lipid/water interface, as reported in the literature.Greater antioxidant activity is found when RSV is embedded in glycosylated liposomes, rather than DOPC/CHOL liposomes. This finding suggests a different RSV distribution in the lipid membrane enclosing the glycosylated amphiphiles, which favor an external exposure of RSV.Biological assays carried out to monitor the demolition effect of RSV-loaded liposomes on mature biofilm of MRSA show that the presence of cationic glycoamphiphiles is essential for a demolition effect to take place on the biofilm matrix. In particular, RSV-galactosylated liposomes are the most effective in destroying MRSA biofilm even at a RSV concentration (0.019 mM) sixty times lower than the MIC (1.2 mM). This work demonstrates, for the first time, how the functionalization of liposomes with cationic glycosydic residues can enhance liposome performances as QSI nanocarriers for the treatment of biofilm associated infections.

Crystal Structures of a Melibiose Symporter MelB with Bound Sugar Substrate

MelB is a well-studied symporter from the major facilitator superfamily of transporters, and its mammalian homologues play important roles in the delivery of essential omega-3 fatty acids into brain and eyes. The bacterial MelB is selective for galactose or galactosides, and catalyzes the obligatory cotransport of the sugar with Na+, Li+, or H+. Previous 3-D crystal structure of the MelBSt of Salmonella thyphimurium shows that MelB adopts a typical MFS-fold, and the thermodynamic ligand binding studies reveal that cooperative binding of Na+ and melibiose is the core mechanism for the obligatory symport. To gain insights into the substrate binding, we solved three sugar-bound crystal structures from two MelBSt mutants that are compromised on Na+ binding. The structures with bound nitrophenyl-α-D-galactoside or melibiose-based detergent dodecyl-β-d-melibioside (DDMB) were refined to a resolution between 3.05 - 3.25 Å. All three structures are nearly identical at an outward-facing conformation; each one contains a á-galactoside molecule in the middle of protein. The binding interactions of galactosyl moiety from each ligand are indistinguishable, revealing a selection pocket, and the non-galactosyl moieties occupy differently. The active site displays a strong salt-bridge/hydrogen-bonding network that provides multiple polar interactions to each of four OH groups on the galactosyl moiety, and the galactosyl ring is sandwiched by two aromatic sidechains via aromatic stacking interactions. There is little or no interaction toward the non-galactosyl moieties, and the entire 12-carbon tail of DDMB lies within the internal cavity. The localized interactions towards the specific galactosyl moiety and the large internal cavity allow MelB to accommodate a variety of galactosides with different structures. The proposed Na+-binding pocket with no bound Na+ seems to lie in a close proximity to the bound sugar. These structures represent a Na+-free, galactoside-bound binary complex of MelB.

Turn on fluorescence sensor for specific detection of glycoligand-receptor recognitions based graphene oxide nanocomposite

We have developed a galactosyl amino-naphthalimide based graphene oxide nanocomposite FN@GO that uses a unique mechanism: Förster resonance energy-transfer (FRET) controlled by interaction between ligands and receptors. The fluorophore stacking to surface of graphene oxide leads to a fluorescence quenching due to FRET. The galactosyl moiety of dye is a glycoligand that selectively recognized by the specific glycoprotein receptors on the surface of liver cancer cells. Therefore, our nanocomposite can provide a concise method to selectively probe HepG2 in cellular environment.

Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB.

β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme’s surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme’s active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthβDG or other analogous galactosidases belonging to GH2 family.

Heparanase-Dependent Remodeling of Initial Lymphatic Glycocalyx Regulates Tissue-Fluid Drainage During Acute Inflammation in vivo.

The glycocalyx is a dense layer of carbohydrate chains involved in numerous and fundamental biological processes, such as cellular and tissue homeostasis, inflammation and disease development. Composed of membrane-bound glycoproteins, sulfated proteoglycans and glycosaminoglycan side-chains, this structure is particularly essential for blood vascular barrier functions and leukocyte diapedesis. Interestingly, whilst the glycocalyx of blood vascular endothelium has been extensively studied, little is known about the composition and function of this glycan layer present on tissue-associated lymphatic vessels (LVs). Here, we applied confocal microscopy to characterize the composition of endothelial glycocalyx of initial lymphatic capillaries in murine cremaster muscles during homeostatic and inflamed conditions using an anti-heparan sulfate (HS) antibody and a panel of lectins recognizing different glycan moieties of the glycocalyx. Our data show the presence of HS, α-D-galactosyl moieties, α2,3-linked sialic acids and, to a lesser extent, N-Acetylglucosamine moieties. A similar expression profile was also observed for LVs of mouse and human skins. Interestingly, inflammation of mouse cremaster tissues or ear skin as induced by TNF-stimulation induced a rapid (within 16 h) remodeling of the LV glycocalyx, as observed by reduced expression of HS and galactosyl moieties, whilst levels of α2,3-linked sialic acids remains unchanged. Furthermore, whilst this response was associated with neutrophil recruitment from the blood circulation and their migration into tissue-associated LVs, specific neutrophil depletion did not impact LV glycocalyx remodeling. Mechanistically, treatment with a non-anticoagulant heparanase inhibitor suppressed LV HS degradation without impacting neutrophil migration into LVs. Interestingly however, inhibition of glycocalyx degradation reduced the capacity of initial LVs to drain interstitial fluid during acute inflammation. Collectively, our data suggest that rapid remodeling of endothelial glycocalyx of tissue-associated LVs supports drainage of fluid and macromolecules but has no role in regulating neutrophil trafficking out of inflamed tissues via initial LVs.

Effects of Enzyme Replacement Therapy and Antidrug Antibodies in Patients with Fabry Disease.

Fabry disease (FD) is an X-linked lysosomal storage disorder (LSD) caused by mutations of the α-galactosidase A gene. The lysosomal enzyme α -galactosidase A (GLA) mediates the hydrolysis of the terminal α -galactosyl moiety from globotriaosylceramide (Gb3). An enzymatic defect leads to the

Synthesis of Galactooligosaccharides in Milk and Whey: A Review.

Galactooligosaccharides (GOS) are synthesized by the enzyme β-galactosidase during the hydrolysis of lactose. In this so-called transgalactosylation reaction the galactosyl moiety is transferred to another sugar molecule instead of water resulting in oligosaccharides of different chain lengths and glycosidic linkages. Because their structures are similar to oligosaccharides present in human breast milk, they act as prebiotics, which has been shown for infants and adults to be alike. While so far most of the research to maximize GOS yield has been carried out using buffered lactose solution as a starting material, more and more work is now conducted with dairy by-products such as whey and whey permeate, or even milk, for direct GOS synthesis in order to develop new GOS-enriched dairy products. This review aims to summarize the results obtained with various dairy liquids, and it rates their suitabilities to act as raw material for GOS production. Most of the studies using whey or milk have been carried out with enzymes from Aspergillus oryzae, Kluyveromyces lactis, Bacillus circulans, Streptococcus thermophilus, and several Lactobacillus species. As the initial lactose concentration (ILC) is known to be a crucial factor for high GOS yield, most of the research has been done with concentrated or supplemented milk and whey. However, a clear dependency on ILC could only be observed for the A. oryzae lactase, indicating a strong influence of milk components like minerals and proteins on the transfer activities of most enzymes.

Isolation, purification, and characterization of AgUCGalT1, a galactosyltransferase involved in anthocyanin galactosylation in purple celery (Apium graveolens L.).

This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins. In the present study, LC-MS data indicated that abundant cyanidin-based anthocyanins accumulated in the petioles of purple celery ('Nanxuan liuhe purple celery'). A gene encoding UDP-galactose: cyanidin 3-O-galactosyltransferase, namely AgUCGalT1, was isolated from purple celery and expressed in Escherichia coli BL21 (DE3). Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase (PSPG) motif. The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment. The recombinant AgUCGalT1 had an optimal enzyme activity at 35°C and pH 8.0, and showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances, namely, peonidin, quercetin, and kaempferol. The expression levels of AgUCGalT1 were positively correlated with the total anthocyanin contents in purple and non-purple celery varieties. Crude enzymes extracted from purple celery exhibited glycosylation ability, whereas crude enzymes obtained from non-purple celery did not have this ability. This work provided evidence as a basis for investigations on the function of AgUCGalT1 in anthocyanin glycosylation in purple celery.

GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors.

Although it has been shown that some mannose-binding lectins (MBLs) exhibit significant activity against HIV infection, little is known about whether N-acetylgalactosamine (GalNAc)-binding lectins have the ability to inhibit HIV infection. Here, we demonstrate that a soybean-derived lectin (SBL) with GalNAc-binding affinity could potently suppress HIV infection of macrophages in a dose-dependent fashion. Unlike the MBLs, which block HIV only through binding to the glycosylated envelope proteins (gp120 and gp41) of the virus, SBL inhibited HIV at multiple steps of the virus infection/replication cycle. SBL could activate the beta interferon (IFN-β)-STAT signaling pathway, resulting in the upregulation of a number of antiviral interferon-stimulated genes (ISGs) in macrophages. In addition, SBL treatment of macrophages induced the production of C-C chemokines, which bind to HIV entry coreceptor CCR5. Deglycosylation of cell surface galactosyl moieties or presaturation of GalNAc-binding capacity could compromise SBL-mediated induction of the antiviral factors. Furthermore, SBL exerted its anti-HIV activity in the low nanomolar range with no mitogenic effect on CD4+ T cells, a major advantage in the development of SBL as a potential anti-HIV agent compared with MBLs. These data indicate a necessity to further investigate SBL as an alternative and cost-effective anti-HIV natural product.IMPORTANCE Mannose-binding lectins (MBLs) can block the attachment of HIV to target cells and have been suggested as anti-HIV microbicides. However, the mitogenic effect of MBLs on CD4+ T cells limits this potential in clinical settings. Lectins with galactose (Gal)- or N-acetylgalactosamine (GalNAc)-binding specificity are another important category of carbohydrate-binding proteins (CBP). Compared to high-mannose N-linked glycans, GalNAc-type glycans present much less in HIV gp120 or gp41 glycosylation. Here, we demonstrate that GalNAc-specific soybean lectin (SBL) triggers antiviral signaling via recognition of the cell surface galactosyl group of macrophages, which results in the suppression of HIV at multiple steps. More importantly, SBL has no mitogenic effect on the activation of CD4+ T cells, a major advantage in the development of Gal/GalNAc-specific lectins as naturopathic anti-HIV agents.

Comparison of Oxidative Stability of Monogalactosyl Diacylglycerol, Digalactosyl Diacylglycerol, and Triacylglycerol Containing Polyunsaturated Fatty Acids

Oxidative stability of three different lipid classes, namely, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach and edible brown seaweed (Akamoku) and triacylglycerol (TAG) of linseed oil was compared. Analysis of oxygen consumption and polyunsaturated fatty acid (PUFA) composition demonstrated that spinach DGDG had the highest oxidative stability, followed by Akamoku DGDG, Akamoku MGDG, spinach MGDG, and linseed TAG. These results disagree with the order of oxidative stability expected from the average number of bis-allylic positions of each lipid. Additionally, DGDG constituents of both spinach and Akamoku showed higher oxidative stability than their MGDG constituents. The unusual oxidative stability of MGDG and DGDG could be conferred by the protection of bis-allylic positions of the PUFA against oxidative attack by the galactosyl moiety of the GL.

Application of Transgalactosylation Activity of \u03b2-Galactosidase from Kluyveromyces lactis for the Synthesis of Ascorbic Acid Galactoside

In view of a commonly known beneficial role and low stability of ascorbic acid, many efforts are constantly undertaken to produce its improved derivatives. This paper presents results on the synthesis of ascorbic acid galactoside using transgalactosylation properties of β-galactosidase from Kluyveromyces lactis and lactose as a donor of galactosyl moiety. The purpose of this study was to determine the influence of selected factors (concentration and molar ratio of substrates, amount of the enzyme preparation, pH of the solution, presence of different ions) on the course of transgalactosylation reaction. Research has shown that approx. 2.5% dry matter (d.m.; 12.7 g/L) of ascorbic acid galactoside is formed under favourable conditions (50% (w/v) substrates, sodium ascorbate and lactose at the molar ratio of 1.9:1, enzyme dose of 28,600 U/100 g lactose, pH = 7.0). The addition of Mg2+ or K+ ions to the reaction medium caused an increase in the final product content (even up to approx. 3.4% d.m., 17.2 g/L), while Na+ or Mn2+ had an adverse impact on the yield. The gathered data may be valuable for cosmetic or food industry.