Thomas Erwin Haselhorst

Carbocycles related to oseltamivir as influenza virus group-1-specific neuraminidase inhibitors. Binding to N1 enzymes in the context of virus-like particles.

We report here the exploitation of the 150-cavity in the active sites of group-1 neuraminidases for the design of new triazole-containing carbocycles related to oseltamivir. Inhibition studies with virus-like particles (VLPs) containing the influenza virus neuraminidase-1 (N1) activity indicate that several candidates are inhibitors, with K(i) values in the 10(-5)-10(-8) M range. In contrast, a known candidate that preserves the free amino group and a new candidate containing a guanidine function are better inhibitors, with K(i) values of 1.5 × 10(-9) and 4.6 × 10(-10) M, respectively. The most active inhibitor of the N1 enzyme in the triazole series was selective for the N1 class and showed significantly less inhibition (K(i) = 2.6 μM vs 0.07 μM) of the free influenza virus neuraminidase-2 (N2). In addition, saturation transfer difference (STD) NMR spectroscopic studies with this compound and the VLPs show that the entire molecule forms contacts with residues in the active site. These data taken together support our proposed binding mode in which the active site and the adjoining 150-cavity are both occupied.

Avian Influenza H5-Containing Virus-Like Particles (VLPs): Host-Cell Receptor Specificity by STD NMR Spectroscopy†

H5N1 influenza virus is a highly contagious pathogen infecting poultry and other birds. The emergence of a human pandemic influenza virus from an avian progenitor appears to involve a switch in preferential binding of the influenza virus hemagglutinin (HA) from a(2,3)-linked N-acetylneuraminic acid containing glycans (the major form in the avian enteric tract) to a(2,6)-linked N-acetylneuraminic acid containing glycans (the major form in the human upper respiratory tract). Avian influenza viruses such as the H3 virus HA receptor typically contain the amino acids Gln226 and Gly228, which create a narrow binding pocket favoring a(2,3)-linked N-acetylneuraminic acid containing glycans. A number of other studies have also investigated HA–glycan recognition. Interestingly, the X-ray crystal structure of the reassembled HA of the 1918 Spanish influenza virus shows that its avianlike narrow binding pocket still allows highaffinity binding of a(2,6)-linked N-acetylneuraminic acid containing glycans. The mutation of only one amino acid (Asp190) within the HA binding site appears to determine the preference of the avianlike 1918 virus for human a(2,6)linked N-acetylneuraminic acid containing glycans. A mutation of Asp190 to Glu190 in the HA of the H5N1 strain could potentially switch its binding preference to a(2,6)linked glycans and consequently result in the emergence of a human pandemic virus. This fact emphasizes an urgent need to investigate the Nacetylneuraminic acid containing glycan recognition requirements by influenza virus HA that may lead to the development of novel antiinfluenza drugs that bind to the viral HA protein and consequently prevent the entry of the virus into human host cells. Rapid access to structural information would provide a detailed understanding of how virus particles interact with host cells on a molecular level and the determinants that prevent interspecies transmission of influenza viruses. Herein we present the first saturation transfer difference (STD) NMR spectroscopic study analyzing H5containing virus-like particles (VLPs) derived from the highly pathogenic avian H5N1 influenza strain in a complex with a(2,6)and a(2,3)-linked N-acetylneuraminides to mimic an in vitro or in vivo virus–host-cell interaction. We recently reported the production of H5 pseudotyped virus particles. To provide larger quantities of suitable VLPs for NMR studies, we have now successfully engineered heterologous H5 influenza VLPs by coexpression of pCDNA–synH5 coding for the hemagglutinin (H5) of H5N1 influenza virus and pCMV-dR8.91-expressing HIV-Gag-pol protein. Coexpression leads to spontaneous assembly of chimeric H5-VLPs, which contain the HA protein. The hemagglutinin, detected as uncleaved HA-0 precursor and HA-1/HA-2 cleaved mature forms, was incorporated on the surface of the viral particles at high levels. Expression of the viral HA protein was characterized by Western blot using specific C-terminal flag-tag M2 antibodies (Figure 1) and detection of VLPs by electron microscopy (Figure 2). In this study we investigated the capacity of H5-VLPs for competitive selection from a mixture of a(2,6)and a(2,3)sialyllactose (6’-SL and 3’-SL, respectively) of a preferred ligand (and therefore linkage) by means of STD NMR spectroscopy. It has been previously demonstrated that STD NMR spectroscopy can be utilized to investigate ligand interactions with whole virus particles, platelets, and intact cells. The large size of viruses and cells makes them attractive for studies with STD NMR spectroscopy because the inherently large line width enables saturation of the particle without affecting ligand signals. Additionally, the larger correlation time of bulky virus particles results in efficient spin diffusion and consequently stronger saturation transfer. To ensure the stability of the influenza H5-VLPs, NMR experiments were performed without prior purification, and 10% D2O was added for locking purposes. The H NMR spectrum of the influenza H5-VLPs (Figure 3a) shows the signals of the 20% sucrose cushion [*] Dr. T. Haselhorst, Dr. T. Islam, F. J. Rose, Prof. M. von Itzstein Institute for Glycomics, Griffith University, Gold Coast Campus Queensland, 4222 (Australia) Fax: (+61)7-555-29040 E-mail: m.vonitzstein@griffith.edu.au Homepage: http://www.griffith.edu.au/glycomics Dr. J.-M. Garcia, J. C. C. Lai, Prof. J. S. M. Peiris Hong Kong University Pasteur Research Centre Ltd., Dexter H.C. Man Building 8 Sassoon Road, Pokfulam, Hong Kong (China)

Leishmania UDP-sugar Pyrophosphorylase. The missing link in galactose salvage?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy

The dense glycocalyx surrounding the protozoan parasite Leishmania is an essential virulence factor. It protects the parasite from hostile environments in the sandfly vector and mammalian host and supports steps of development and invasion. Therefore, new therapeutic concepts concentrate on disturbing glycocalyx biosynthesis. Deletion of genes involved in the metabolism of galactose and mannose have been shown to drastically reduce Leishmania virulence. Here we report the identification of Leishmania major UDP-glucose pyrophosphorylase (UGP). UGP catalyzes the formation of UDP-glucose from glucose 1-phosphate and UTP. This activation step enables glucose to enter metabolic pathways and is crucial for the activation of galactose. UDP-galactose is made from UDP-glucose by nucleotide-donor transfer to galactose 1-phosphate or by epimerization of the glucose moiety. Isolated in a complementation cloning approach, the activity of L. major UGP was proven in vitro. Moreover, purified protein was used to investigate enzyme kinetics, quaternary organization, and binding of ligands. Whereas sequestration by oligomerization is a known regulatory mechanism for eukaryotic UGPs, the recombinant as well as native L. major UGP migrated as monomer in size exclusion chromatography and in accord with this showed simple Michaelis-Menten kinetics toward all substrates. In saturation transfer difference (STD)-NMR studies, we clearly demonstrated that the molecular geometry at position 4 of glucose is responsible for substrate specificity. Furthermore, the γ-phosphate group of UTP is essential for binding and for induction of the open conformation, which then allows entry of glucose 1-phosphate. Our data provide the first direct proof for the ordered bi-bi mechanism suggested in earlier studies.

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.

Revisiting the role of histo-blood group antigens in rotavirus host-cell invasion

Histo-blood group antigens (HBGAs) have been proposed as rotavirus receptors. H type-1 and Lewis(b) antigens have been reported to bind VP8* from major human rotavirus genotypes P[4], P[6] and P[8], while VP8* from a rarer P[14] rotavirus recognizes A-type HBGAs. However, the role and significance of HBGA receptors in rotavirus pathogenesis remains uncertain. Here we report that P[14] rotavirus HAL1166 and the related P[9] human rotavirus K8 bind to A-type HBGAs, although neither virus engages the HBGA-specific α1,2-linked fucose moiety. Notably, human rotaviruses DS-1 (P[4]) and RV-3 (P[6]) also use A-type HBGAs for infection, with fucose involvement. However, human P[8] rotavirus Wa does not recognize A-type HBGAs. Furthermore, the common human rotaviruses that we have investigated do not use Lewis(b) and H type-1 antigens. Our results indicate that A-type HBGAs are receptors for human rotaviruses, although rotavirus strains vary in their ability to recognize these antigens.

Structure and function of nucleotide sugar transporters: Current progress

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure–function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Recognition of the GM3 Ganglioside Glycan by Rhesus Rotavirus Particles

Rotaviruses are a major cause of severe infantile gastroenteritis in humans and animals worldwide, producing a childhood mortality exceeding 650 000 annually.[1] Mapping host cell glycan-virus interactions to define a viral glycointeractome is invaluable in providing new directions for the discovery of novel broad-spectrum drugs and vaccines. In that context we have recently reported the first NMR-based structural analysis of the interaction of GD1a (1) and GM1 (2) ganglioside glycans with recombinantly expressed rotaviral surface lectin VP8* from two distinct rotavirus strains.[2]

Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2β1 integrin in mediating rotavirus infection.

ABSTRACT N-acetyl- and N-glycolylneuraminic acids (Sia) and α2β1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2β1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2β1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCE Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan deficient (cgl) plants

There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, half-maximal inhibitory concentration of isofagomine and thermal stability of the cgl-derived GCase were comparable with those of imiglucerase, a commercially available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose terminated. Additional purification was required to remove ∼15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including the use of a null cgl mutant.