Tailoring a novel sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein by natural evolution-mimicry

Sialic acid (Sia) is a typical terminal sugar, which modifies various types of glycoconjugates commonly found in higher animals. Its regulatory roles in diverse biological phenomena are frequently triggered by interaction with Sia-binding lectins. When using natural Sia-binding lectins as probes, however, there have been practical problems concerning their repertoire and availability. Here, we show a rational creation of a Sia-binding lectin based on the strategy 'natural evolution-mimicry', where Sia-binding lectins are engineered by error-prone PCR from a Gal-binding lectin used as a scaffold protein. After selection with fetuin-agarose using a recently reinforced ribosome display system, one of the evolved mutants SRC showed substantial affinity for alpha2-6Sia, which the parental Gal-binding lectin EW29Ch lacked. SRC was found to have additional practical advantages in productivity and in preservation of affinity for Gal. Thus, the developed novel Sia-recognition protein will contribute as useful tools to sialoglycomics.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins

Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.     

Investigating the effects of point mutations on the affinity between the cyanobacterial lectin microvirin and high mannose-type glycans present on the HIV envelope glycoprotein

Human immunodeficiency virus (HIV) infections continue to exert an enormous impact on global human health. This led experts to emphasize the importance of new measures for preventing HIV infections, including the development of vaccines and novel drugs. In this context, a promising approach involves the use of lectins that can bind the surface envelope glycoprotein gp120 of HIV with high affinity, preventing viral entry. The cyanobacterial lectin microvirin (MVN) has been proposed as a candidate for development as a topical microbicide because of its ability to bind to high mannose-type glycans, potently inhibiting HIV-1 entry. Thus, the aim of this computational study was to investigate the effects of four point mutations (D53Q, D53E, D53K, and D53W) on the structure and affinity of MVN with di-mannose (MAN). Molecular dynamics simulations followed by binding free energy calculations using MM-GBSA were employed. The calculated binding free energy of ligand-receptor complexation of MVN with MAN was ?26.02 kcal mol^-1. We identified in the wild-type protein that residues I45, T59, and Q81 have a major contribution to the binding free energy of di-mannose. Among the investigated mutants, the most promising one was the D53W mutation, with a theoretical binding free energy value of ?29.16 kcal mol^-1. We suggest that this increased stability is due to the introduction of extra rigidity on the hinge region connecting two key structural elements of the MVN binding site.     

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.     

Mucin-lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.     

Glycomic mapping of <Emphasis Type="Italic">O-</Emphasis> and <Emphasis Type="Italic">N-</Emphasis>linked glycans from major rat sublingual mucin

Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galβ1→4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.     

Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin

Lectins belonging to the jacalin-related lectin family are distributed widely in the plant kingdom. Recently, two mannose-specific lectins having tandem repeat-type structures were discovered in Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm). The occurrence of such similar molecules in taxonomically less related plants suggests their importance in the plant body. To obtain clues to understand their physiological roles, we performed detailed analysis of their sugar-binding specificity. For this purpose, we compared the dissociation constants (K(d)) of Castanea crenata agglutinin (CCA) and Cycas revoluta leaf lectin (CRLL) by using 102 pyridylaminated and 13 p-nitrophenyl oligosaccharides with a recently developed automated system for frontal affinity chromatography. As a result, we found that the basic carbohydrate-binding properties of CCA and CRLL were similar, but differed in their preference for larger N-linked glycans (e.g. Man7-9 glycans). While the affinity of CCA decreased with an increase in the number of extended alpha1-2 mannose residues, CRLL could recognize these Man7-9 glycans with much enhanced affinity. Notably, both lectins also preserved considerable affinity for mono-antennary, complex type N-linked glycans, though the specificity was much broader for CCA. The information obtained here should be helpful for understanding their functions in vivo as well as for development of useful probes for animal cells. This is the first systematic approach to elucidate the fine specificities of plant lectins by means of high-throughput, automated frontal affinity chromatography.     

Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein

Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced widely enough (at least 32 A) that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.     

Mutation of aspartate 804 of Na(+),K(+)-ATPase modifies the cation binding pocket and thereby generates a high Na(+)-ATPase activity

A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.     

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of α1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of α1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K_d) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for α1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K_a) for α1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were α1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of α1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.     

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.     

Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo.

We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with prolonged simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the alpha1-6 arm and stacking of the bisecting GlcNAc and the neighboring Man/GlcNAc residues of both antennae. Chemoenzymatic synthesis produced the bisecting biantennary decasaccharide N-glycan and its alpha2-3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity alterations in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and animals. The panel of lectins, including an adhesion/growth-regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [André, S., Unverzagt, C., Kojima, S., Dong, X., Fink, C., Kayser, K. & Gabius, H.-J. (1997) Bioconjugate Chem. 8, 845-855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injection of iodinated neoglycoproteins carrying galactose-terminated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications.     

A structural determinant of the unique interfacial binding mode of bovine pancreatic phospholipase A2.

The catalytic steps of the phospholipase A2 (PLA2)-catalyzed hydrolysis of phospholipids are preceded by interfacial binding. Among various pancreatic PLA2s, bovine pancreatic PLA2 (bpPLA2) has a unique interfacial binding mode in which Lys-56 plays an important role in its binding to anionic lipid surfaces. To identify the structural determinant of this unique interfacial binding mode of bpPLA2, we systematically mutated bpPLA2 and measured the effects of mutations on its interfacial binding and activity. First, different cationic clusters were generated in the amino-terminal alpha-helix by the N6R, G7K, and N6R/G7K mutations. These mutations enhanced the binding of bpPLA2 to anionic liposomes up to 15-fold. For these mutants, however, the K56E mutation still caused a large drop in interfacial affinity for and activity toward anionic liposomes, indicating that the generation of a cationic patch in the amino-terminal alpha-helix of bpPLA2 did not change its interfacial binding mode. Second, residues 62-66 that form a part of the pancreatic loop were deleted. For this deletion mutant (Delta62-66), which was as active as wild-type toward anionic liposomes, the K56E and K116E mutations (Delta62-66/K56E and Delta62-66/K116E) did not have significant effects on interfacial affinity. In contrast, the K10E mutation showed a much larger decrease in interfacial affinity (10-fold), indicating the deletion of residues 62-66 caused a major change in the interfacial binding mode. Finally, hydrophobic residues in positions 63 and 65 were replaced by bulkier ones (V63F and V63F/V65L) to pinpoint the structural determinant of the interfacial binding mode of bpPLA2. The effects of K10E and K56E mutations on the interfacial affinity and activity of these mutants showed that Val-63 and Val-65 of bpPLA2 are the structural determinant of its unique interfacial binding mode and that relatively conservative substitutions at these positions result in large changes in the interfacial binding mode among mammalian pancreatic PLA2s. Taken together, this study reveals how minor structural differences among homologous PLA2s can lead to distinct interfacial binding behaviors.     

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the fungal pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. The independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW structure, water loss, and cuticle deposition. Furthermore, 138 F2 plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.     

表面电荷突变与胰蛋白酶底物专一性的改造

分别利用酶切重组和“３＋１”引物ＰＣＲ定点突变的方法构建了三个胰蛋白酶表面电荷双突变体：Ｒ６２Ｄ＋Ｋ９７Ｅ、Ｒ６２Ｄ＋Ｋ１７５Ｅ和Ｋ９７Ｅ＋Ｋ１７５Ｅ．对三者在Ｅ．ｃｏｌｉＸ－９０菌中的表达产物进行了动力学测定,分别得到了三种双突变体在两种ｐＨ条件下,水解ＴＡＭＥ、ＴＬＭＥ两种底物的动力学数据．结果表明,Ｒ６２Ｄ＋Ｋ１７５Ｅ和Ｋ９７Ｅ＋Ｋ１７５Ｅ在ｐＨ６．８５时,对两种底物的催化活性与野生型相比下降了２～３个数量级,当ｐＨ升高至８．８５时,它们的活性基本丧失;双突变体Ｒ６２Ｄ＋Ｋ９７Ｅ虽然催化活性也有所降低,但随着ｐＨ的升高,它对Ｌｙｓ底物的特异性（选择性系数２５倍于Ａｒｇ底物）转变为对Ａｒｇ底物略高的特异性,基本符合分子设计．实验结果还表明,各种双突变体催化活性的降低主要是由于酶和底物的亲和力降低引起的．     

Diverse sugar-binding specificities of marine invertebrate C-type lectins

The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized alpha2-6 sialylation but not alpha2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with alpha-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis(x) and Lewis(a) epitopes.     

Replacement of several single amino acid side chains exposed to the inside of the ATP-binding pocket induces different extents of affinity change in the high and low affinity ATP-binding sites of rat Na/K-ATPase

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na(+)/K(+)-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na(+)/K(+)-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na(+)/K(+)-ATPase or low affinity ATP inhibition of K(+)-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K(0.5). However, both the affinities for the K480E were reduced to approximately 1/20. The turnover number of the Na(+)/K(+)-ATPase and the apparent affinity for Na(+) and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K(+)-pNPPase and the apparent affinity for K(+) were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.