Enzymatic Sialylation of Synthetic Multivalent Scaffolds: From 3'-Sialyllactose Glycomacromolecules to Novel Neoglycosides.

Sialoglycans play a key role in many biological recognition processes and sialylated conjugates of various types have successfully been applied, e.g., as antivirals or in antitumor therapy. A key feature for high affinity binding of such conjugates is the multivalent presentation of sialoglycans which often possess synthetic challenges. Here, the combination is described of solid phase polymer synthesis and enzymatic sialylation yielding 3'-sialyllactose-presenting precision glycomacromolecules. CMP-Neu5Ac synthetase from Neisseria meningitidis (NmCSS) and sialyltransferase from Pasteurella multocida (PmST1) are combined in a one-pot reaction giving access to sequence-defined sialylated macromolecules. Surprisingly, when employing Tris(hydroxymethyl)aminomethane (Tris) as a buffer, formation of significant amounts of α-linked Tris-sialoside is observed as a side reaction. Further exploring and exploiting this unusual sialylation reaction, different neoglycosidic structures are synthesized showing that PmST1 can be used to derive both, sialylation on natural carbohydrates as well as on synthetic hydroxylated scaffolds.

NMR Experiments Shed New Light on Glycan Recognition by Human and Murine Norovirus Capsid Proteins.

Glycan-protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.

Fragment Growing to Design Optimized Inhibitors for Human Blood Group†B Galactosyltransferase (GTB).

Human blood group B galactosyltransferase (GTB) catalyzes the galactosylation of the H antigen and is responsible for the formation of the blood group antigen of phenotype B. The ABO blood group system is well studied and routinely serotyped before transfusion and transplantation. Blood type subgroups have been repeatedly linked to an increased occurrence of diseases (e.g., a highly increased incidence rate for pancreatic cancer for individuals with blood group phenotype B). 3-Phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole 1 has previously been described to inhibit GTB with a Ki value of 800 µm. In this work, we describe a computer-guided fragment-growing approach for the optimization of this fragment that was subsequently realized by synthesizing the most promising ligands. Enlarging the phenyl moiety of fragment 1 to a naphthyl moiety resulted in ligand 3-(naphthalene-1-yl)-5-(piperazin-1-yl)-1,2,4-thiadiazole 2 a, which shows a threefold improvement in binding affinity (Ki =271 µm).

Functional binding of hexanucleotides to 3C protease of hepatitis A virus.

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal ß-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.

Binding of an acceptor substrate analog enhances the enzymatic activity of human blood group B galactosyltransferase.

The hydrolysis of the donor substrate uridine diphosphate galactose (UDP-Gal) by human blood group B galactosyltransferase (GTB) has been followed by nuclear magnetic resonance in the presence and in the absence of an acceptor substrate analog. It is observed that the presence of the acceptor substrate analog promotes hydrolysis of UDP-Gal. Subsequent analysis of the kinetics of the enzymatic hydrolysis suggests that this effect is due to an increased affinity of GTB for UDP-Gal in the presence of the acceptor analog. Isothermal titration calorimetry experiments substantiate this conclusion. As hydrolysis may be understood as a glycosyl transfer reaction where water serves as universal acceptor, we suggest that in general the binding of acceptor substrates to retaining glycosyltransferases modulates the rate of glycosyl transfer. In fact, this may point to a general mechanism used by retaining glycosyltransferases to discriminate acceptor substrates under physiological conditions.

NMR analysis of carbohydrate-protein interactions.

Carbohydrate-protein interactions are frequently characterized by dissociation constants in the microM to mM range. This is normally associated with fast dissociation rates of the corresponding complexes, in turn leading to fast exchange on the nuclear magnetic resonance (NMR) chemical shift time scale and on the NMR relaxation time scale. Therefore, NMR experiments that take advantage of fast exchange are well suited to study carbohydrate-protein interactions. In general, it is possible to analyze ligand binding by observing either protein signals or ligand resonances. Because most receptor proteins to which carbohydrates bind are rather large with molecular weights significantly exceeding 30 kDa, the analysis of the corresponding protein spectra is not trivial, and only very few studies have been addressing this issue so far. We, therefore, focus on NMR experiments that employ observation of free ligand, that is, carbohydrate signals to analyze the bound state. Two types of NMR experiments have been extremely valuable to analyze carbohydrate-protein interactions at atomic resolution. Whereas transferred nuclear Overhauser effect (NOE) experiments deliver bioactive conformations of carbohydrates binding to proteins, saturation transfer difference (STD) NMR spectra provide binding epitopes and valuable information about the binding thermodynamics and kinetics. We demonstrate the power of a combined transfer NOE/STD NMR approach for the analysis of carbohydrate-protein complexes using selected examples.

Blood group B galactosyltransferase: insights into substrate binding from NMR experiments.

The biosynthesis of human blood group B antigens is accomplished by a highly specific galactosyltransferase (GTB). On the basis of NMR experiments, we propose a "molecular tweezers mechanism" that accounts for the exquisite stereoselectivity of donor substrate selection. Transferred NOE experiments for the first time reveal the bioactive conformation of the donor substrate UDP-galactose (UDP-Gal) and of its enzymatically inactive analogue, UDP-glucose (UDP-Glc). Both bind to GTB in a folded conformation that is sparsely populated in solution, whereas acceptor ligands bind in a conformation that predominates in solution. The bound conformations of UDP-Gal and UDP-Glc are identical within experimental error. Therefore, GTB must discriminate between the two activated sugars on the basis of a hitherto unknown transition state that can only be formed in the case of UDP-Gal. A full relaxation and exchange matrix analysis of STD NMR experiments reveals that acceptor substrates dissociate significantly faster (k(off) > 100 Hz) from the binding pocket than donor substrates (k(off) approximately 10 Hz). STD NMR experiments also directly show that proper recognition of the hexopyranose rings of the UDP sugars requires bivalent metal cations. At the same time, this analysis furnishes the complete three-dimensional structure of the enzyme with its bound donor substrate UDP-Gal on the basis of a prior crystal structure analysis. We propose that, upon acceptor binding, GTB uses the Asp 302 and Glu 303 side chains as "molecular tweezers" to promote bound UDP-Gal but not UDP-Glc into a transition state that leads to product formation.

Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR.

Saturation transfer difference NMR experiments on human blood group B alpha-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and beta-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that beta-D-galactose and alpha-D-galactose bind weakly to GTB. Whereas beta-D-galactose binds to the acceptor and donor sites, it is suggested that alpha-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg(2+) and Mn(2+) is more relevant under physiological conditions.

Assaying sialyltransferase activity with surface plasmon resonance.

Here, we describe an activity assay for sialyltransferases based on surface plasmon resonance (SPR). Different natural and synthetic oligosaccharides serving as acceptor substrates for the sialyltransferase ST3Gal-III (EC 2.4.99.6) were immobilized or synthesized on SPR chips. The chip was then exposed to different concentrations of a reaction mixture of ST3Gal-III and CMP-Neu5Ac either by injection or by external application of the reaction mixture to the chip surface. The binding of two lectins, one that specifically recognizes the unmodified acceptor, the other the sialylated oligosaccharide, was utilized to determine the extent of enzymatic turnover. In order to obtain enzymatic activities, the SPR data were correlated to data obtained from a classical radio assay. After regeneration, that is, cleavage of the sialic acid residues by using a sialidase, the chip is available for new experiments. The technique allows the rapid determination of sialyltransferase activity with only nanomolar quantities of acceptor substrates and should be of particular value in cases in which a large variety of samples, including cell lysates, have to be screened for their enzymatic activities.

Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization.

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils.

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.