Affinity For Sugars Research Articles

Role of the irreplaceable residues in the LacY alternating access mechanism

Few side chains in the galactoside/H(+) symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H(+) translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H(+) symport is abolished as well.

Jatropha curcas hemagglutinin is similar to a 2S albumin allergen from the same source and has unique sugar affinities

We have previously reported the purification and preliminary X-ray characterization of a hemagglutinin from the seeds of Jatropha curcas and, with the detailed sequencing information available now, we find that it is similar to a 2S albumin allergen isolated from the same source. Through a search of Jatropha genome database (http://www.kazusa.or.jp/jatropha/), we map it to the sequence id JcCA0234191 (now referred to as Jcr4S00619.70 in the new version, release 4.5) which has a conserved alpha amylase inhibitor/seed storage protein domain found in the 2S albumin allergens. The putative sequence of the small and large chains of the protein is assigned and the total mass of the two subunits matches with the intact mass 10 kDa determined through MALDI. The protein retains hemagglutination activity between pH 6-9 and up to 60 °C on heat treatment and its hemagglutination activity is inhibited by sialic acid and fetuin. Bioinformatics studies show that the isolated protein sequence clusters in close association with a 2S albumin from Ricinus communis in phylogeny analysis and has a conservation of the characteristic four disulfide linkage pattern. Hemagglutinins and lectins are known to have allergenic effects through their interaction with immunoglobulin E and histamine release and earlier studies have shown that this interaction can be inhibited by lectin-specific sugars. We hope this report bridges the plant allergens and hemagglutinins further for exploring possible mediation of allergenic activity through sialic acid and complex sugar interactions and generates further interest in the area.

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon(Decapoda, Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)-labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti-FITC-conjugated gold-lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar-associated components of the HE. This revealed that both Con A and WGA-binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold-labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N-acetyl-β-d-glucosamine (sugar affinity to WGA).

Activity and Safety of Synthetic Lectins Based on Benzoboroxole-Functionalized Polymers for Inhibition of HIV Entry

Lectins derived from plant and microbial sources constitute a vital class of entry inhibitors that target the oligomannose residues on the HIV envelope gp120. Despite their potency and specificity, success of lectin-based entry inhibitors may be impeded by high manufacturing costs, formulation and potential mitogenicity. Therefore, there exists a gap in the HIV microbicides pipeline that underscores the need for mass producible, synthetic, broad-spectrum, and biocomptabile inhibitors of HIV entry. Here, we present the development of a polymeric synthetic lectin, based on benzoboroxole (BzB), which exhibits weak affinity (∼25 M(-1)) for nonreducing sugars, similar to those found on the HIV envelope. High molecular weight BzB-functionalized polymers demonstrated antiviral activity that increased with an increase in ligand density and molecular weight of the polymer construct, revealing that polyvalency improves activity. Polymers showed significant increase in activity from 25 to 75 mol % BzB functionalization with EC(50) of 15 μM and 15 nM, respectively. A further increase in mole functionalization to 90% resulted in an increase of the EC(50) (59 ± 5 nM). An increase in molecular weight of the polymer at 50 mol % BzB functionalization showed a gradual but significant increase in antiviral activity, with the highest activity seen with the 382 kDa polymer (EC(50) of 1.1 ± 0.5 nM in CEM cells and 11 ± 3 nM in TZM-bl cells). Supplementing the polymer backbone with 10 mol % sulfonic acid not only increased the aqueous solubility of the polymers by at least 50-fold but also demonstrated a synergistic increase in anti-HIV activity (4.0 ± 1.5 nM in TZM-bl cells), possibly due to electrostatic interactions between the negatively charged polymer backbone and the positively charged V3-loop in the gp120. The benzoboroxole-sulfonic acid copolymers showed no decrease in activity in the presence of a seminal concentration of fructose (p > 0.05). Additionally, the copolymers exhibit minimal, if any, effect on the cellular viability, barrier properties, or cytokine levels in human reconstructed ectocervical tissue after 3 days of repeated exposure and did not show pronounced activity against a variety of other RNA and DNA viruses.

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin

The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77-G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady-state model for the kinetically controlled synthesis of galacto-oligosaccharides (GOS) with Aspergillus oryzae β-galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta-oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl-enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl-enzyme complex is higher than that of the free enzyme.

Sugar Binding Residue Affects Apparent Na+ Affinity and Transport Stoichiometry in Mouse Sodium/Glucose Cotransporter Type 3B

SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.

Evaluation of in vitro gas production and rumen bacterial populations fermenting corn milling (co)products

The objective of this study was to evaluate the fermentation dynamics of 2 commonly fed corn (co)products in their intact and defatted forms, using the in vitro gas production (IVGP) technique, and to investigate the shifts of the predominant rumen bacterial populations using the 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) technique. The bTEFAP technique was used to determine the bacterial profile of each fermentation time at 24 and 48h. Bacterial populations were identified at the species level. Species were grouped by substrate affinities (guilds) for cellulose, hemicellulose, pectin, starch, sugars, protein, lipids, and lactate. The 2 (co)products were a dried distillers grain (DDG) plus solubles produced from a low-heat drying process (BPX) and a high-protein DDG without solubles (HP). Chemical analysis revealed that BPX contained about 11.4% ether extract, whereas HP contained only 3.88%. Previous studies have indicated that processing methods, as well as fat content, of corn (co)products directly affect fermentation rate and substrate availability, but little information is available regarding changes in rumen bacterial populations. Fermentation profiles of intact and defatted BPX and HP were compared with alfalfa hay as a standard profile. Defatting before incubation had no effect on total gas production in BPX or HP, but reduced lag time and the fractional rate of fermentation of BPX by at least half, whereas there was no effect for HP. The HP feed supported a greater percentage of fibrolytic and proteolytic bacteria than did BPX. Defatting both DDG increased the fibrolytic (26.8 to 38.7%) and proteolytic (26.1 to 37.2%) bacterial guild populations and decreased the lactate-utilizing bacterial guild (3.06 to 1.44%). Information regarding the fermentation kinetics and bacterial population shifts when feeding corn (co)products may lead to more innovative processing methods that improve feed quality (e.g., deoiling) and consequently allow greater inclusion rates in dairy cow rations.

Differential effects of sucrose and fructose on dietary obesity in four mouse strains

We examined sugar-induced obesity in mouse strains polymorphic for Tas1r3, a gene that codes for the T1R3 sugar taste receptor. The T1R3 receptor in the FVB and B6 strains has a higher affinity for sugars than that in the AKR and 129P3 strains. In Experiment 1, mice had 40 days of access to lab chow plus water, sucrose (10 or 34%), or fructose (10 or 34%) solutions. The strains consumed more of the sucrose than isocaloric fructose solutions. The pattern of strain differences in caloric intake from the 10% sugar solutions was FVB > 129P3 = B6 > AKR; and that from the 34% sugar solutions was FVB > 129P3 > B6 ≥ AKR. Despite consuming more sugar calories, the FVB mice resisted obesity altogether. The AKR and 129P3 mice became obese exclusively on the 34% sucrose diet, while the B6 mice did so on the 34% sucrose and 34% fructose diets. In Experiment 2, we compared total caloric intake from diets containing chow versus chow plus 34% sucrose. All strains consumed between 11 and 25% more calories from the sucrose-supplemented diet. In Experiment 3, we compared the oral acceptability of the sucrose and fructose solutions, using lick tests. All strains licked more avidly for the 10% sucrose solutions. The results indicate that in mice (a) Tas1r3 genotype does not predict sugar-induced hyperphagia or obesity; (b) sucrose solutions stimulate higher daily intakes than isocaloric fructose solutions; and (c) susceptibility to sugar-induced obesity varies with strain, sugar concentration and sugar type.

A Single Amino Acid Change Converts the Sugar Sensor SGLT3 into a Sugar Transporter

BackgroundSodium-glucose cotransporter proteins (SGLT) belong to the SLC5A family, characterized by the cotransport of Na+ with solute. SGLT1 is responsible for intestinal glucose absorption. Until recently the only role described for SGLT proteins was to transport sugar with Na+. However, human SGLT3 (hSGLT3) does not transport sugar but causes depolarization of the plasma membrane when expressed in Xenopus oocytes. For this reason SGLT3 was suggested to be a sugar sensor rather than a transporter. Despite 70% amino acid identity between hSGLT3 and hSGLT1, their sugar transport, apparent sugar affinities, and sugar specificity differ greatly. Residue 457 is important for the function of SGLT1 and mutation at this position in hSGLT1 causes glucose-galactose malabsorption. Moreover, the crystal structure of vibrio SGLT reveals that the residue corresponding to 457 interacts directly with the sugar molecule. We thus wondered if this residue could account for some of the functional differences between SGLT1 and SGLT3.Methodology/Principal FindingsWe mutated the glutamate at position 457 in hSGLT3 to glutamine, the amino acid present in all SGLT1 proteins, and characterized the mutant. Surprisingly, we found that E457Q-hSGLT3 transported sugar, had the same stoichiometry as SGLT1, and that the sugar specificity and apparent affinities for most sugars were similar to hSGLT1. We also show that SGLT3 functions as a sugar sensor in a living organism. We expressed hSGLT3 and E457Q-hSGLT3 in C. elegans sensory neurons and found that animals sensed glucose in an hSGLT3-dependent manner.Conclusions/SignificanceIn summary, we demonstrate that hSGLT3 functions as a sugar sensor in vivo and that mutating a single amino acid converts this sugar sensor into a sugar transporter similar to SGLT1.

Functional characterization of mouse sodium/glucose transporter type 3b

Despite belonging to a family of sugar cotransporters, human sodium/glucose transporter type 3 (hSGLT3) does not transport sugar, but it depolarizes the cell in the presence of extracellular sugar, and thus it has been suggested to work as a sugar sensor. In the human genome there is one SGLT3 gene, yet in mouse there are two. In this study we cloned one of them, mouse SGLT3b (mSGLT3b) and characterized the protein. We found that mSGLT3b has low affinity for sugars, as does hSGLT3, but surprisingly, mSGLT3b transports sugar, although the sugar transport is not as tightly coupled to cations as in SGLT1. Moreover, the sugar specificity of mSGLT3b has characteristics reminiscent of both SGLT1 and hSGLT3: mSGLT3b does not respond to galactose, similar to hSGLT3, but neither does it respond to 1-deoxynojirimycin, unlike hSGLT3 but similar to SGLT1. mSGLT3b has low apparent affinities for sugar and Na(+) and, furthermore, displays pre-steady-state currents, which in SGLT1 report on conformational changes in the protein. Finally, phlorizin, the typical inhibitor of SGLT proteins, also inhibits mSGLT3b. In summary, although mSGLT3b has some characteristics that resemble SGLT1 and others that are similar to hSGLT3, its low sugar affinity and uncoupled sugar transport lead us to conclude that mSGLT3b likely functions as a physiological glucose sensor similar to hSGLT3.

A new mistletoe Phthirusa pyrifolia leaf lectin with antimicrobial properties

Phthirusa pyrifolia leaf lectin (PpyLL) was obtained from the hemi-parasitic medicinal plant mistletoe through saline saturation and two consecutive chromatographic steps on Sephadex G-100 and ion exchange on CM-cellulose. SDS-PAGE of the protein under non-reducing conditions revealed a monomeric protein with a molecular weight of 15.6 kDa. Under reducing conditions in the presence of 2-mercaptoethanol, the protein showed two bands with molecular weights of 15 and 7 kDa. PpyLL, an acidic glycoprotein with 19% sugar content, was not dependent on divalent cations. It was stable up to 70 °C and exhibited maximum hemagglutination at pH 7.5. Lectin fluorescence emission spectra at different temperatures showed that the lectin fluorescence increased when the temperature increased. PpyLL showed a unique affinity for the phosphate derivative of fructose, fructose-1-6-biphosphate. PpyLL showed effective antimicrobial activities against bacteria (Gram-positive: Staphylococcus epidermidis, Streptococcus faecalis and Bacillus subtilis; Gram-negative: Klebsiella pneumoniae) and fungi ( Fusarium lateritium and Rhizoctonia solani). Therefore, PpyLL specificity, as determined by a new sugar affinity, may be significant to determine its biological potential.

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack. The nucleotide sequence reported in this paper has been submitted to the GenBank nucleotide database under accession number AM981401.

Properties of a LacY Efflux Mutant

Crystal structures of lactose permease from Escherichia coli (LacY) exhibit two six-helix bundles with 2-fold pseudosymmetry separated by a large hydrophilic cavity. The cavity is open only on the cytoplasmic side and contains the side chains important for both sugar and H(+) binding at the apex in the middle of the protein; the periplasmic side is tightly closed. A plethora of biochemical and biophysical data strongly support an alternating access mechanism in which both the sugar- and H(+)-binding sites are exposed alternatively to either side of the membrane by reciprocal opening and closing of cytoplasmic and periplasmic cavities. Here we describe a unique mutation that results in an increase in sugar efflux. Asp240 (helix VII), which interacts with Lys319 (helix X), also comprises part of a salt-bridge/H-bond network that is critically involved in the mechanism of sugar/H(+) symport. The mutant, which contains Glu in place of Asp240, exhibits a marked decrease in active lactose transport and an enhanced rate of downhill lactose/H(+) efflux. Transport is increased to normal levels when the sugar concentration is increased 10-fold, consistent with the decrease in sugar affinity observed for this mutant. Taken as a whole, the results suggest that the primary defect induced by the mutation may involve a decrease in affinity for H(+).

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K(d)(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K(d)(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH on K(d)(app) inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.

Protonation and sugar binding to LacY

The effect of bulk-phase pH on the apparent affinity (K(d)(app)) of purified wild-type lactose permease (LacY) for sugars was studied. K(d)(app) values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K(d)(app) for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pK(a) of approximately 10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H(+) symport in either direction across the membrane.

Morphological and glycohistochemical studies on the epididymal region of the Sudani duck ( Cairina moschata)

In this study, the epididymal region of the Sudani duck was investigated using histological and lectin histochemical methods. Morphologically, the epididymal region of the Sudani duck is composed of extratesticular rete testis, proximal and distal efferent ductules, a short connecting duct, and epididymal ducts. Morphometric analysis of the epididymal region of Sudani duck revealed that the efferent ductules predominate in relation to the epididymal ducts. The distribution of sugar moieties within the epididymal region of the Sudani duck was investigated using ten different fluorescein isothiocyanate (FITC) conjugated lectins. In the rete testis epithelium, only PHA-L showed a positive reaction. Efferent ductules in contrary exhibited a wide range of lectin affinity whereas six positive lectins (Con A, LCA, PNA, WGA, PHA-L, PHA-E) were observed. In the connecting and epididymal ducts, four lectins (Con A, WGA, PHA-L, PHA-E) were also detected. GSA-I, UEA-I, and LTA were at all not evident in the epididymal region of the Sudani duck. In conclusion, the correlation between the large areas of the epididymal region occupied by the efferent ductules and the wide range of sugar affinity of this portion may confirm the speculation that efferent ductules might be the primary site of fluid reabsorption in the epididymal region of Sudani duck.

Galactose binds to focal segmental glomerulosclerosis permeability factor and inhibits its activity

Focal segmental glomerulosclerosis (FSGS) is associated with circulating permeability activity (Palb) and recurs after transplantation in about 30% of patients. The FS permeability factor (FSPF) consists of anionic low-molecular-weight protein(s) that might be excluded by the anionic filtration barrier. We postulated that FSPF may interact with sugars of the glycocalyx, and we tested its affinity for sugars using column chromatography. FSPF showed high affinity for galactose; Palb activity was absent from unbound material and present in eluate after dialysis to remove galactose. In parallel studies, Palb activity of serum was lost after adding galactose > or = 10(-12) M. To determine whether galactose also abolishes plasma Palb activity in vivo, a patient with posttransplant FSGS was given galactose and serum samples were collected. Intravenous infusion of galactose decreased Palb from 0.88 before infusion to undetectable levels postinfusion and at 48 hours. Oral galactose diminished Palb activity; Palb reached a nadir after 2 weeks and remained low for at least 4 weeks after galactose was discontinued. We conclude that FSPF has high affinity for galactose based on chromatography. Additionally, galactose inactivates FSPF and may lead to its clearance from plasma. The interaction between FSPF and glomeruli may depend on FSPF binding to galactose, and the FSPF-galactose complex may be susceptible to uptake by galactose-binding proteins and to catabolism. We propose testing galactose as a novel nontoxic therapy for nephrotic syndrome in FSGS to determine whether galactose slows progression and whether pretransplant therapy decreases rates of recurrence and graft loss.

Dissecting carbohydrate–Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition

The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.

Crosslinking of low-affinity glycoprotein ligands to galectin LEC-1 using a photoactivatable sulfhydryl reagent

The N-terminal lectin domain (Nh) of the tandem repeat-type nematode galectin LEC-1 has a lower affinity for sugars than the C-terminal lectin domain. To confirm that LEC-1 forms a complex with N-acetyllactosamine-containing glycoproteins, we used several mutants of LEC-1 in which a unique cysteine residue was introduced into the Nh domain and examined their binding to bovine asialofetuin with a photoactivatable sulfhydryl crosslinking reagent. A crosslinked product was formed with the Q38C mutant, strongly suggesting the low-affinity interaction of Nh with the glycoprotein could be detected with this system.