Chemoenzymatic synthesis of N-acetyl-D-neuraminic acid from N-acetyl-D-glucosamine by using the spore surface-displayed N-acetyl-D-neuraminic acid aldolase

Chemoenzymatic synthesis of N-acetyl-d-neuraminic acid from N-acetyl-d-glucosamine using the spore surface-displayed N-acetyl-d-neuraminic acid aldolase at a high concentration (53.9 g liter(-1)) was achieved in this study. Thus, displaying a target enzyme on the surface of spores might be an alternative for integration of biocatalytic conversion into chemical synthesis.     

Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.     

Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects

In this study, we report the first cloning and characterization of a N-acetylneuraminic acid phosphate synthase gene from Drosophila melanogaster, an insect in the protostome lineage. The gene is ubiquitously expressed at all stages of Drosophila development and in Schneider cells. Similar to the human homologue, the gene encodes an enzyme with dual substrate specificity that can use either N-acetylmannosamine 6-phosphate or mannose 6-phosphate to generate phosphorylated forms of both the sialic acids, N-acetylneuraminic acid and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, respectively, when expressed in either bacterial or baculoviral expression systems. The identification of a functional sialic acid synthase in Drosophila indicates that insects have the biosynthetic capability to produce sialic acids endogenously. Although sialylation is widely distributed in organisms of the deuterstome lineage, genetic evidence concerning the presence or absence of sialic acid metabolism in organisms of the protostome lineage has been lacking. Homology searches of the Drosophila genome identified putative orthologues of other genes required for sialylation of glycoconjugates.     

Detection of the Neu5 Ac (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3- and alpha 2,6-linkage

The Maackia amurensis leukoagglutinin has been shown to react specifically with the Neu5Ac (alpha 2,3) Gal sequence of asparagine-linked complex type oligosaccharides. We report here the preparation of Maackia amurensis lectin-gold complexes and their application for light and electron microscopic detection of the Neu5 Ac (alpha 2,3) Gal sequence in various tissues. The use of the lectin directly gold labeled was superior to a two-step cytochemical affinity technique using a fetuin-gold complex. The Maackia amurensis lectin-gold staining was inhibited by pre-incubation of the lectin-gold complexes with 50 mM alpha 2,3 sialyllactose, whereas alpha 2,6 sialyllactose up to concentrations of 1 M had no effect, thus demonstrating the high specificity of the histochemical staining. In addition to N-glycanase-sensitive asparagine-linked oligosaccharides, beta-elimination-sensitive serine/threonine-linked oligosaccharides could be detected. Data are presented which show that cellular staining patterns obtained with Maackia amurensis lectin-gold complexes may differ from those with elderberry bark lectin-gold, which detects the Neu5 Ac (alpha 2,6) Gal/GalN Ac sequence. Electron microscopic double labeling for direct study of the differential distribution of the Neu5 Ac (alpha 2,3) Gal and Neu5 Ac (alpha 2,6) Gal sequences is reported. Therefore, the availability of two sialic acid binding lectins with different linkage specificity for histochemistry provides the first opportunity to study tissue and cell type expression of these terminal sequences of glycoproteins.     

Large scale purification of gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides G_M3(Neu5Ac) and G_M3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure G_M3(Neu5Ac) and G_M3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of G_M3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two G_M3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl- versus N-glycolylneuraminic acid).     

Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked beta 1-2, beta 1-4, or beta 1-6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.     

N-乙酰神经氨酸在离子交换树脂AD-1上的吸附热/动力学

主要研究了N-乙酰神经氨酸(Neu5Ac)在AD-1离子交换树脂上的吸附行为。通过静态摇瓶的方法,利用Langmuir模型对热力学平衡数据进行拟合,测得树脂的最大Neu5Ac吸附量为0.39 g/g,且不受温度影响。依次用大孔扩散模型、大孔微孔扩散模型对不同Neu5Ac初始质量浓度的动力学数据进行拟合,结果表明,在较低浓度和较高浓度下,Neu5Ac在AD-1树脂上的离子交换过程的限速步骤依次为大孔扩散和大孔微孔扩散,其中大孔扩散系数Dp为1.453×10-8m2/min,微孔扩散系数Dc为9.153×10-15m2/min。最后利用大孔微孔扩散模型研究了树脂颗粒度大小对Neu5Ac在AD-1树脂上的离子交换动力学的影响,发现选取较小颗粒度的树脂有利于提高离子交换速率,且模型与实验数据吻合较好,从而验证了模型的准确性。AD-1树脂对Neu5Ac有较大的吸附容量和较快的吸附速率。     

Purification and identification of N-glycolylneuraminic acid (Neu5Gc) from the holothuroidea Gumi, Cucumaria echinata.

N-Glycolylneuraminic acid (Neu5Gc), precious sialic acid which could not be synthesized by a chemical method, occurrs in the body of holothuroidea, Gumi Cucumaria echinata. Gumi contains 85% of total sialic acid, as Neu5Gc, in the body. Neu5Gc was purified from dry powder of the body using Dowex 1-x8 (HCOO* form) anion exchange chromatography after mild acid hydrolysis with 0.1 N trifluoroacetic acid. Using GC-MS and 1H-NMR spectroscopy, the purified Neu5Gc was correctly identified to be Neu5Gc. The purity of Neu5Gc was more than 99%. This is the first report of purification and identification of Neu5Gc from holothuroidea by using anion exchange chromatography, GC-MS, and 1H-NMR.     

LC-MS/MS法检测猪内脏和肌肉中N-乙酰神经氨酸、N-羟乙酰氨酸和脱氨神经氨酸浓度的研究

目的:建立液相色谱-三重四级杆质谱联用法(LC-MS/MS)测定猪内脏和肌肉组织中唾液酸N-乙酰神经氨酸(Neu5Ac)、N-羟乙酰神经氨酸(Neu5Gc)和脱氨神经氨酸(KDN)浓度的分析方法。方法:将内脏肉组织匀浆后,利用三氟乙酸(TFA)将结合态的唾液酸从糖脂或糖蛋白链上解离下来后进样分析。色谱条件:色谱柱为ZORBAX Eclipse Plus C18(5μm,4.6×250 mm,Waters),流动相为0.1%醋酸铵-100%乙腈(9:1),柱温:25℃,流速800μL·min~(-1),进样量20μL。质谱条件:离子源为电喷雾化离子源(ESI),扫描方式为多重反应监测(MRM),监测离子对:Neu5Ac:307.7→87.0,Neu5Gc:323.3→116.0,KDN:266.7→87.0,13C3Neu5Ac内标品:310.9→90.0。结果:Neu5Ac、Neu5Gc和KDN分别在0.1~20.0、0.05~10.00和0.005~1.000μmol·L~(-1)的浓度范围内和对照品与内标峰面积比值线性关系良好,重复性平均RSD为1.2%,稳定性平均RSD为1.9%,日间和日内精密度试验RSD均小于6.7%,平均回收率为92.9~106.4%。结论:本方法简便、快速、灵敏度高,可广泛运用于组织和体液中唾液酸的测定。     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc

We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.     

High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature

The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of Neu5Ac with alternating alpha(2-8) and alpha(2-9) linkages. It accumulates when the bacterium is grown at 37 degrees C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 micrograms x ml-1) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. The enzyme, CMP-Neu5Ac synthetase, was not detected in cells grown at 20 degrees C. The lack of this enzyme explains the absence of polymer biosynthesis when the bacterium was grown at 20 degrees C.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6)Gal/GalNAc sequence

Carbohydrate binding properties of a new plant lectin isolated from elderberry (Sambucus nigra L.) (SNA) bark were studied using the techniques of quantitative precipitation, hapten inhibition, and equilibrium dialysis. Purified SNA precipitates highly sialylated glycoproteins such as fetuin, orosomucoid, and ovine submaxillary mucin, but not their asialo derivatives. Hapten inhibition experiments showed that both D-Gal and D-GalNAc are weak inhibitors of SNA-glycophorin precipitation, but neither New5Ac nor Neu5Gc is an inhibitor. A series of oligosaccharides which contain the terminal Neu5Ac(alpha 2-6)Gal sequence showed an extremely high inhibitory potency (1,600-10,000 times more inhibitory than Gal). On the other hand, oligosaccharides with the Neu5Ac(alpha 2-3)Gal linkage were only 30-80 times more inhibitory than Gal, thus showing a marked preference for the 2,6-linked isomer. Hapten inhibition with Gal and its epimers suggested that the equatorial OH at C-3 and the axial OH at C-4 of the D-pyranose ring are strict requirements for binding. Conversion of the Neu5Ac residue to its 7-carbon analogue by selective periodate oxidation of its glyceryl side chain, followed by NaBH4 reduction, completely destroyed the ability of fetuin and orosomucoid to precipitate with SNA. Moreover, the same treatment of Neu5Ac(alpha 2-3)lactitol also abolished its ability to inhibit the precipitation reaction, suggesting that the glyceryl side chain of NBu5Ac (especially the C-8 and/or C-9 portion) is an important determinant for SNA. The increased inhibitory potency of various glycosides with beta-linked nonpolar aglycons suggested the presence of a hydrophibic interacting region adjacent to the carbohydrate binding site. The results of equilibrium dialysis using [3H] Neu5Ac(alpha 2-6)lactitol as ligand showed the presence of two equivalent, noninteracting carbohydrate binding sites in this tetrameric glycoprotein lectin (Ka = 3.9 X 10(5) M-1).     

Enzymatic biocatalysis in chemical synthesis of pharmaceuticals (Review)

In this review we summarize the available research on enzymatic biocatalysis in the chemical synthesis of drugs. We focus on oxydoreductsases, particularly ketoreductases, that are widely used in biotechnological processes: alpha- and omega-transaminases, lipases, nitrile hydrolases, and aldolases. The potential for the extended use of novel enzymes produced via bioengineering is discussed.     

Controlled-release biocatalysis for the synthesis of D-phenylglycine

An isolated and immobilised aminotransferase cloned from Pseudomonas stutzeri ST-201 into Escherichia coli was used to synthesise d-phenylglycine. The reaction was characterised by an unfavourable equilibrium constant and substrate inhibition. The use of a controlled-release system via the use of Amberlite (IRA 400)-adsorbed benzoylformate proved a useful technique to circumvent these issues. This resulted in a four-fold improvement in product concentration achievable to yield a final d-phenylglycine concentration of 10.25 mg/ml.     

Synthesis of Neu5Ac(alpha2-->6)[Gal(alpha1-->3)]GalNAcalpha and Neu5Ac(alpha2-->6)[Gal(beta1-->3)]GalNAcalpha trisaccharides as protected trifluoroacetamidopropyl glycosides

Protected sialo-containing trisaccharides, fragments of oligosaccharide chains of mucin glycoproteins, were synthesized. Regioselective sialylation of the primary hydroxyl group of (3-fluoroacetamidopropyl)-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-ben zyl)-alpha -D-galactopyranosyl)-alpha-D-galactopyranoside with methyl ester of peracetyl-beta-ethylthioglycoside of N-acetylneuraminic acid in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) yielded 39 and 25% of alpha- and beta-sialyl-(2-->6)biosides, respectively. Catalytic hydrogenolysis of the azide and benzyl groups of the alpha-anomer followed by N- and O-acetylation gave target trifluoroacetamidopropyl glycoside, Neu5Ac(alpha 2-->6)[Gal(alpha 1-->3)]GalNAc alpha-OSp, as a peracetate. An analogous coupling of the sialyl donor with (3-fluoroacetamidopropyl)-2-acetamido-2-deoxy-3-O- (2,3,4,6-tetra-O-acetyl)-beta-D-galactopyranosyl)-alpha-D-galactopyranos ide affords acetylated trifluoroacetamidopropyl glycoside Neu5Ac(alpha 2-->6)[Gal(beta 1-->3)]GalNAc alpha-OSp in a yield of 15% and the corresponding Neu5Ac(beta 2-->6)-anomer in a yield of 12%. After O-deacetylation and N-detrifluoroacetylation, these sialylbiosides can be used as ligands in preparing neoglycoconjugates.     

Synthesis of Neu5Ac- and Neu5Gc-alpha-(2-->6')-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-alpha-(2-->6')-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-alpha-(2-->6')-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4',6'-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

A direct synthesis of pyrrolocytosine from 5-iodocytosine

We have employed a tandem Sonogashira/annulation reaction between 5-iodocytosine derivatives and terminal alkynes to yield the fluorescent bicyclic nucleobase pyrrolcytosine. Pyrrolocytosine bearing substituents only on the pyrrole ring are conveniently synthesized from 5-iodocytosine. Water soluble pyrrolocytosines are being investigated as reporter groups in SNP analysis.     

Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Ac{alpha}2-6Gal{beta}1-4GlcNAc human-type influenza receptor

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7??) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding.