Group B Streptococcus (GBS) is classified into nine serotypes that vary in capsular polysaccharide (CPS) architecture but share in common the presence of a terminal sialic acid (Sia) residue. This position and linkage of GBS Sia closely resembles that of cell surface glycans found abundantly on human cells. CD33-related Siglecs (CD33rSiglecs) are a family of Sia-binding lectins expressed on host leukocytes that engage host Sia-capped glycans and send signals that dampen inflammatory gene activation. We hypothesized that GBS evolved to display CPS Sia as a form of molecular mimicry limiting the activation of an effective innate immune response. In this study, we applied a panel of immunologic and cell-based assays to demonstrate that GBS of several serotypes interacts in a Sia- and serotype-specific manner with certain human CD33rSiglecs, including hSiglec-9 and hSiglec-5 expressed on neutrophils and monocytes. Modification of GBS CPS Sia by O acetylation has recently been recognized, and we further show that the degree of O acetylation can markedly affect the interaction between GBS and hSiglec-5, -7, and -9. Thus, production of Sia-capped bacterial polysaccharide capsules that mimic human cell surface glycans in order to engage CD33rSiglecs may be an example of a previously unrecognized bacterial mechanism of leukocyte manipulation.

Carbohydrate antigen sialyl Lewis a (CA19-9) is the most frequently applied serum tumor marker for diagnosis of cancers in the digestive organs. Recent progress disclosed the presence of a normal counterpart of the determinant, namely disialyl Lewis a, which is predominantly expressed in non-malignant epithelial cells of the digestive organs, while sialyl Lewis a is preferentially expressed in cancers. The disialyl Lewis a determinant carries one extra sialic residue attached through a 2 ->> 6 linkage to the GlcNAc moiety compared to cancer-associated sialyl Lewis a, which carries only one 2 ->> 3 linked sialic acid residue (monosialyl Lewis a). Disialyl Lewis a in normal epithelial cells serves as a ligand for immunosuppressive receptors such as sialic acid binding immunoglobulin (Ig)-like lectins (siglec-7) and -9 expressed on resident monocytes/macrophages and maintains immunological homeostasis of mucosal membranes in digestive organs. Epigenetic silencing of a gene for a 2 ->> 6 sialyl-transferase in the early stages of carcinogenesis results in an impairment of 2 ->> 6 sialylation, leading to incomplete synthesis and accumulation of sialyl Lewis a, which lacks the 2 ->> 6 linked sialic acid residue, in cancer cells. Simultaneous determination of serum levels of sialyl- and disialyl Lewis a, and calculation of the monosialyl/disialyl Lewis a ratio provide information useful for excluding a false-positive serum diagnosis, and also for averting the undesired influence of the Lewis blood group of patients on serum antigen levels. During the course of cancer progression in locally advanced cancers, tumor hypoxia induces transcription of several glycogenes involved in sialyl Lewis a synthesis. Expression of the determinant, consequently, is further accelerated in more malignant hypoxia-resistant cancer cell clones, which become predominant clones in advanced stage cancers and frequently develop hematogenous metastasis. Sialyl Lewis a, as well as its positional isomer sialyl Lewis x, serves as a ligand for vascular cell adhesion molecule E-selectin and facilitates hematogenous metastasis through mediating adhesion of circulating cancer cells to vascular endothelium. Patients having both strong sialyl Lewis a expression on cancer cells and enhanced E-selectin expression on vascular beds are at a greater risk of developing distant hematogenous metastasis.

BACKGROUND

Sialic acid, a key component of both human milk oligosaccharides and neural tissues, may be a conditional nutrient during periods of rapid brain growth.

OBJECTIVE

We tested the hypothesis that variations in the sialic acid content of a formula milk would influence early learning behavior and gene expression of enzymes involved in sialic acid metabolism in piglets.

METHODS

Piglets (n = 54) were allocated to 1 of 4 groups fed sow milk replacer supplemented with increasing amounts of sialic acid as casein glycomacropeptide for 35 d. Learning performance and memory were assessed with the use of easy and difficult visual cues in an 8-arm radial maze. Brain ganglioside and sialoprotein concentrations and mRNA expression of 2 learning-associated genes (ST8SIA4 and GNE) were measured.

RESULTS

In both tests, the supplemented groups learned in significantly fewer trials than did the control group, with a dose-response relation for the difficult task (P = 0.018) but not the easy task. In the hippocampus, significant dose-response relations were observed between amount of sialic acid supplementation and mRNA levels of ST8SIA4 (P = 0.002) and GNE (P = 0.004), corresponding with proportionate increases in protein-bound sialic acid concentrations in the frontal cortex.

CONCLUSIONS

Feeding a protein-bound source of sialic acid during early development enhanced learning and increased expression of 2 genes associated with learning in developing piglets. Sialic acid in mammalian milks could play a role in cognitive development.

Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a triple beta-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis.

The nature of the receptors for mucoid and nonmucoid Pseudomonas aeruginosa was investigated by using adherence to injured tracheal epithelium as a model. Bovine submaxillary mucin and crude rat tracheal mucin inhibited the adherence of both types of P. aeruginosa. Among the sugars present in these mucins only N-aceylneuraminic acid inhibited adherence. Inhibition of adherence probably involved the binding of N-acetylneuraminic acid to the bacterial cells and not to the tracheal cells. The mucoid strain appeared to be much more sensitive to inhibition by N-acetylneuraminic acid. Periodate oxidation and cholera filtrate also reduced the adherence of both strains, but Clostridium perfringens neuraminidase treatment did not alter adherence. A nonmucoid isogenic mutant of an unstable mucoid strain was also inhibited by N-acetylneuraminic acid. These data suggest that the receptor for P. aeruginosa is a sialic acid moiety on cell surfaces or in mucins.

The B lymphocyte cell surface receptor CD22 is an adhesion molecule that can mediate binding to several leukocyte subsets. The first CD22 ligand to be identified was the receptor-linked phosphotyrosine phosphatase CD45, but several lines of evidence suggest that CD22 may interact with multiple counter receptors on adjacent lymphocytes. In the present work, we show that in addition to CD45, a soluble CD22-immunoglobulin fusion protein (CD22Rg) recognizes several other distinct lymphocyte sialoglycoproteins. CD22-mediated adhesion is dependent upon the presence of sialic acids on ligands. CD22Rg is observed to bind specifically to a 115-kDa sialoglycoprotein in COS cells transfected with an alpha-2,6-sialyltransferase cDNA, but not in COS cells transfected with unrelated cDNA clones, indicating that at least some CD22-mediated interactions require presentation of sialic acid in an alpha-2,6 linkage by CD22 ligands. In all cases, truncation of the side chain of sialic acids by mild periodate oxidation abolishes recognition by CD22Rg. Direct binding of CD22Rg to lymphoid cells also requires sialic acids and their side chains. Taken together, these observations indicate that CD22 is a sialic acid-binding lectin and may define a novel functional subset of immunoglobulin superfamily adhesion molecules.

CD22 beta is a glycoprotein found on the surface of B cells during restricted stages of development. It is believed to play a role in cell-cell interactions and B cell activation. The accompanying paper (Sgroi, D., Varki, A., Braesch-Andersen, S., and Stamenkovic, I. (1993) J. Biol. Chem. 268, 7011-7018) shows that CD22 beta recognizes multiple glycoproteins on the surfaces of T and B cells and that sialylation of these ligands is essential for binding. To identify the structure(s) of the sialylated oligosaccharide(s) recognized by CD22 beta, [3H]glucosamine-labeled glycoproteins were purified from Daudi cells by adsorption onto a CD22 beta recombinant immunoglobulin (CD22 beta Rg) chimera attached to protein A-Sepharose (PAS), and the N-linked oligosaccharides were released by peptide N-glycosidase F. These released oligosaccharides failed to bind to CD22 beta Rg-PAS under the conditions used initially to adsorb the glycoproteins, but their elution from a column of CD22 beta Rg-PAS was significantly retarded. Populations of oligosaccharides with different affinities could be identified by their order of elution. Specific sialidases were used to determine the content of alpha-2,3- and alpha-2,6-linked sialic acid in these different populations and their contribution to binding. Multiantennary oligosaccharides with one alpha-2,6-linked residue bound marginally, and those with two or more bound more tightly. alpha-2,3-Linked sialic acid residues were without effect. Binding did not require divalent cations and was abrogated by mild periodate oxidation of the outer side chain of sialic acid. No marked differences in size or fucose content were found between the populations of high and low affinity oligosaccharides. However, the low affinity population could be partially converted into higher affinity by treatment with beta-galactoside alpha-2,6 sialyltransferase and CMP-sialic acid. Thus, CD22 beta is a mammalian lectin that can recognize specific N-linked oligosaccharide structures containing alpha-2,6-linked sialic acids.

Baculovirus expression vectors are frequently used to express glycoproteins, a subclass of proteins that includes many products with therapeutic value. The insect cells that serve as hosts for baculovirus vector infection are capable of transferring oligosaccharide side chains (glycans) to the same sites in recombinant proteins as those that are used for native protein N-glycosylation in mammalian cells. However, while mammalian cells produce compositionally more complex N-glycans containing terminal sialic acids, insect cells mostly produce simpler N-glycans with terminal mannose residues. This structural difference between insect and mammalian N-glycans compromises the in vivo bioactivity of glycoproteins and can potentially induce allergenic reactions in humans. These features obviously compromise the biomedical value of recombinant glycoproteins produced in the baculovirus expression vector system. Thus, much effort has been expended to characterize the potential and limits of N-glycosylation in insect cell systems. Discoveries from this research have led to the engineering of insect N-glycosylation pathways for assembly of mammalian-style glycans on baculovirus-expressed glycoproteins. This chapter summarizes our knowledge of insect N-glycosylation pathways and describes efforts to engineer baculovirus vectors and insect cell lines to overcome the limits of insect cell glycosylation. In addition, we consider other possible strategies for improving glycosylation in insect cells.

Prostate-specific antigen (PSA), the tumor marker currently used for prostate cancer (PCa), is not specific enough to distinguish between PCa and benign prostate hyperplasia (BPH). Glycan processing is normally perturbed in tumors, therefore we investigated whether changes in glycosylation of PSA could be useful diagnostic indicators. Previously we determined that the glycosylation of PSA secreted by the tumor prostate cell line LNCaP differs significantly from that of PSA from seminal plasma (normal control). We therefore undertook a detailed glycan analysis of PSA derived from sera from PCa patients and, importantly, established that the glycosylation of the PCa serum PSA was significantly different from the PSA from the LNCaP cell line. In comparison with seminal plasma PSA, the fucose content of PSA from the PCa patient serum was significantly lower and there was a decrease in alpha2,3-linked sialic acid. Differences in the glycosylation of PSA derived from PCa patients' sera, seminal plasma, and LNCaP cells were further established by lectin detection, glycosylation immunosorbent assay, and two-dimensional electrophoresis. We also investigated whether the impact of glycosylation changes initiated by the tumor was reflected in the serum glycome. By comparing the glycans released from the total glycoproteins in PCa patient serum with those of normal serum we found an increase in the proportion of sialyl-Lewis x structures. Further analysis of the glycosylation of PSA from PCa and BPH sera will be required in order to determine the utility of these glycan differences to discriminate specifically between benign and malignant prostate states.

Galectins are β-galactoside-binding lectins that regulate diverse cell behaviors, including adhesion, migration, proliferation, and apoptosis. Galectins can be expressed both intracellularly and extracellularly, and extracellular galectins mediate their effects by associating with cell-surface oligosaccharides. Despite intensive current interest in galectins, strikingly few studies have focused on a key enzyme that acts to inhibit galectin signaling, namely β-galactoside α2,6-sialyltransferase (ST6Gal-I). ST6Gal-I adds an α2,6-linked sialic acid to the terminal galactose of N-linked glycans, and this modification blocks galectin binding to β-galactosides. This minireview summarizes the evidence suggesting that ST6Gal-I activity serves as an "off switch" for galectin function.

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.

CD22 is a negative regulator of B cell signaling, an activity modulated by its interaction with glycan ligands containing alpha2-6-linked sialic acids. B cells deficient in the enzyme (ST6Gal I) that forms the CD22 ligand show suppressed BCR signaling. Here we report that mice deficient in both CD22 and its ligand (Cd22-/- St6gal1-/- mice) showed restored B cell receptor (BCR) signaling, suggesting that the suppressed signaling of St6gal1-/- cells is mediated through CD22. Coincident with suppressed BCR signaling, B cells lacking ST6Gal I showed a net redistribution of the BCR to clathrin-rich microdomains containing most of the CD22, resulting in a twofold increase in the localization of CD22 together with the BCR. These studies suggest an important function for the CD22-ligand interaction in regulating BCR signaling and microdomain localization.

Site-specific mutations have been made in the influenza hemagglutinin (HA) receptor binding site to assess the contribution of individual amino acid residues to receptor recognition. Screening of mutant HAs, expressed using recombinant vaccinia virus-infected cells, for their abilities to bind human erythrocytes indicated that substitutions involving conserved residues Y98F, H183F, and L194A severely restricted binding and that the substitution W153A prevented cell surface expression of HA. Mutation of residues E190 and S228 that are in positions to form hydrogen bonds with the 9-OH of sialic acid appeared to increase erythrocyte binding slightly, as did the substitution G225R. Substitutions of other residues that are directly or indirectly involved in receptor binding, S136T, S136A, Y195F, G225D, and L226P, had intermediate effects on binding between these two extremes. Estimates of changes in receptor binding specificity based on inhibition of binding to erythrocytes by nonimmune horse sera indicated that mutants G225R and L226P, unlike wild-type HA, were not inhibited; Y195F and G225D mutants were, like wild type, inhibited; and erythrocyte binding by mutants S136A, S136T, E190A, and S228G was only partially inhibited. Viruses containing mutant HAs Y98F, S136T, G225D, and S228G that cover the range of erythrocyte binding properties observed were also constructed by transfection. All four transfectant viruses replicated in MDCK cells and embryonated hens' eggs as efficiently as wild-type X-31 virus, although the Y98F mutant virus was unable to agglutinate erythrocytes. Mutant MDCK cells that have reduced levels of cell surface sialic acids were susceptible to infection by S136T, G225D, and S228G transfectant viruses and by wild type but not by the Y98F transfectant virus.

BACKGROUND

Avian influenza A H6N1 virus is one of the most common viruses isolated from wild and domestic avian species, but human infection with this virus has not been previously reported. We report the clinical presentation, contact, and environmental investigations of a patient infected with this virus, and assess the origin and genetic characteristics of the isolated virus.

METHODS

A 20-year-old woman with an influenza-like illness presented to a hospital with shortness of breath in May, 2013. An unsubtyped influenza A virus was isolated from her throat-swab specimen and was transferred to the Taiwan Centres for Disease Control (CDC) for identification. The medical records were reviewed to assess the clinical presentation. We did a contact and environmental investigation and collected clinical specimens from the case and symptomatic contacts to test for influenza virus. The genomic sequences of the isolated virus were determined and characterised.

RESULTS

The unsubtyped influenza A virus was identified as the H6N1 subtype, based on sequences of the genes encoding haemagglutinin and neuraminidase. The source of infection was not established. Sequence analyses showed that this human isolate was highly homologous to chicken H6N1 viruses in Taiwan and had been generated through interclade reassortment. Notably, the virus had a G228S substitution in the haemagglutinin protein that might increase its affinity for the human α2-6 linked sialic acid receptor.

CONCLUSIONS

This is the first report of human infection with a wild avian influenza A H6N1 virus. A unique clade of H6N1 viruses with a G228S substitution of haemagglutinin have circulated persistently in poultry in Taiwan. These viruses continue to evolve and accumulate changes, increasing the potential risk of human-to-human transmission. Our report highlights the continuous need for preparedness for a pandemic of unpredictable and complex avian influenza.

BACKGROUND

Taiwan Centres for Disease Control.

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Moreover, the virus causes outbreaks in poultry and other animals, such as pigs, requiring costly and laborious countermeasures. Therefore, influenza virus has a substantial impact on health and the global economy. Here, we review entry of this important pathogen into target cells, an essential process by which viral genomes are delivered from extracellular virions to sites of transcription/replication in the cell nucleus. We summarize current knowledge on the interaction of influenza viruses with their receptor, sialic acid, and highlight the ongoing search for additional receptors. We describe receptor-mediated endocytosis and the recently discovered macropinocytosis as alternative virus uptake pathways, and illustrate the subsequent endosomal trafficking of the virus with advanced live microscopy techniques. Release of virus from the endosome and import of the viral ribonucleoproteins into the host cell nucleus are also outlined. Although a focus has been on viral protein function during entry, recent studies have revealed exciting information on cellular factors required for influenza virus entry. We highlight these, and discuss established entry inhibitors targeting viral and host factors, as well as the latest prospects for designing novel 'anti-entry' compounds. New entry inhibitors are of particular importance for current efforts to develop the next generation of anti-influenza drugs - entry is the first essential step of virus replication and is an ideal target to block infection efficiently.

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Human rotaviruses (Wa, KUN, MO) showed hemagglutination (HA) only with fixed 1-day-old chicken erythrocytes, and their HA activities were completely destroyed by trypsin activation of virions. Simian SA-11 and bovine NCDV had HA activities not only against fixed erythrocytes but also against fresh erythrocytes from various species. Their HA activities against fixed erythrocytes were also inhibited by trypsin activation, but those against fresh erythrocytes were not. Neuraminidase treatment of fixed erythrocytes did not inhibit HA by trypsin-untreated rotaviruses. In contrast, HA of fresh human erythrocytes by SA-11 and NCDV was completely inhibited by neuraminidase treatment of erythrocytes or glycophorin A, the major erythrocyte sialoglycoprotein. Adsorption and infection of SA-11 and NCDV to monkey kidney MA104 cells were also inhibited by neuraminidase treatment of cells. Adsorption and infection of human rotaviruses were not, however, affected by treatment of cells with neuraminidase from Vibrio cholerae or Arthrobacter ureafaciens or with potassium periodate. Therefore, HA of fixed chicken erythrocytes by trypsin-untreated human and animal rotaviruses may be independent of sialic acids, whereas that of fresh erythrocytes by SA-11 and NCDV is sialic acid dependent and probably mediated by glycophorin A. Sialic acids also constitute an essential part of the cellular receptors for SA-11 and NCDV, whereas those for human rotaviruses were quite resistant to treatments known to destroy major types of sialic acids.

The sialic acids are major components of the cell surfaces of animals of the deuterostome lineage. Earlier studies suggested that humans may not express N-glycolyl-neuraminic acid (Neu5Gc), a hydroxylated form of the common sialic acid N-acetyl-neuraminic acid (Neu5Ac). We find that while Neu5Gc is essentially undetectable on human plasma proteins and erythrocytes, it is a major component in all the four extant great apes (chimpanzee, bonobo, gorilla and orangutan) as well as in many other mammals. This marked difference is also seen amongst cultured lymphoblastoid cells from humans and great apes, as well as in a variety of other tissues compared between humans and chimpanzees, including the cerebral cortex and the cerebrospinal fluid. Biosynthetically, Neu5Gc arises from the action of a hydroxylase that converts the nucleotide donor CMP-Neu5Ac to CMP-Neu5Gc. This enzymatic activity is present in chimpanzee cells, but not in human cells. However, traces of Neu5Gc occur in some human tissues, and others have reported expression of Neu5Gc in human cancers and fetal tissues. Thus, the enzymatic capacity to express Neu5Gc appears to have been suppressed sometime after the great ape-hominid divergence. As terminal structures on cell surfaces, sialic acids are involved in intercellular cross-talk involving specific vertebrate lectins, as well as in microbe-host recognition involving a wide variety of pathogens. The level of sialic acid hydroxylation (level of Neu5Ac versus Neu5Gc) is known to positively or negatively affect several of these endogenous and exogenous interactions. Thus, there are potential functional consequences of this widespread structural change in humans affecting the surfaces of cells throughout the body.

The biosynthesis and maturation of the oligosaccharide moieties of the envelope glycoprotein of vesicular stomatitis virus were investigated in virus-infected HeLa and BHK21 cells after pulse labeling with [2-3H]mannose. Two major forms of the virus glycoprotein were detected by polyacrylamide gel electrophoresis, which appear to correspond to the viral glycoprotein with either "precursor" or "mature" oligosaccharide chains. The precursor chains in both HeLa and BHK21 cells infected with vesicular stomatitis virus obtained after a 30-min pulse were large oligomannose structures containing approximately 7--9 mannose residues as estimated by gel filtration analysis. The size of the oligomannose structures initially transferred to the protein may have been even larger. Mature, virus-size oligosaccharide chains, which could be detected after a 20- to 30-min delay, contained only three mannose residues and, in addition, contained branch structures terminating in sialic acid. A precursor--product relationship of these two forms of oligosaccharide chains was demonstrated by pulse--chase labeling of virus-infected HeLa cells. These studies indicated that the large oligomannosyl core structures initially added to the glycoprotein were being "trimmed" by the removal of mannose residues prior to (and/or during) the addition of the branch chains terminating in sialic acid.

Among coronaviruses, several members are able to interact with sialic acids. For bovine coronavirus (BCoV) and related viruses, binding to cell surface components containing N-acetyl-9- O-acetylneuraminic acid is essential for initiation of an infection. These viruses resemble influenza C viruses because they share not only the receptor determinant, but also the presence of an acetylesterase that releases the 9- O-acetyl group from sialic acid and thus abolishes the ability of the respective sialoglycoconjugate to function as a receptor for BCoV. As in the case of influenza viruses, the receptor-destroying enzyme of BCoV is believed to facilitate the spread of virus infection by removing receptor determinants from the surface of infected cells and by preventing the formation of virus aggregates. Another coronavirus, porcine transmissible gastroenteritis virus (TGEV) preferentially recognizes N-glycolylneuraminic acid. TGEV does not contain a receptor-destroying enzyme and does not depend on the sialic acid binding activity for infection of cultured cells. However, binding to sialic acids is required for the enteropathogenicity of TGEV. Interaction with sialoglycoconjugates may help the virus to pass through the sialic acid-rich mucus layer that covers the viral target cells in the epithelium of the small intestine. We discuss that the BCoV group of viruses may have evolved from a TGEV-like ancestor by acquiring an acetylesterase gene through heterologous recombination.

Trans-sialidases (TS) are GPI-anchored surface enzymes expressed in specific developmental stages of trypanosome parasites like Trypanosoma cruzi, the etiologic agent of Chagas disease, and T. brucei, the causative agent of sleeping sickness. TS catalyzes the transfer of sialic acid residues from host to parasite glycoconjugates through a transglycosidase reaction that appears to be critical for T. cruzi survival and cell invasion capability. We report here the structure of the T. cruzi trans-sialidase, alone and in complex with sugar ligands. Sialic acid binding is shown to trigger a conformational switch that modulates the affinity for the acceptor substrate and concomitantly creates the conditions for efficient transglycosylation. The structure provides a framework for the structure-based design of novel inhibitors with potential therapeutic applications.

These investigations were directed at furnishing information on the essential structural features of the cold-insoluble globulin of human plasma. Amino acid and carbohydrate analyses showed that it is a glycoprotein (1.2% sialic acid, 1.8% hexose, 2.1% hexosamine) containing all of the amino acids usually found in proteins. Circular dichroic spectral analysis suggested that cold-insoluble globulin contained a very high proportion of beta-structure; no evidence for the presence of alpha-helix was found. Sedimentation velocity experiments at pH 7.0, in the presence or absence of dithiothreitol, plus related gel electrophoretic experiments at pH 8.4, indicated that the integrity of certain disulfide bridges was necessary for its solubility under "physiologic" conditions. In experiments in urea-containing solution, two sedimenting peaks were observed. The major one, amounting to more than 95% of the total, had an s20,w of 5.6 S, the minor peak had an s20,w of 7.3 S. Following disulfide bridge reduction a single symmetrical peak of 3.9 S was formed. Such behavior suggested that cold-insoluble globulin is a multichain molecule whose subunit chains are linked by disulfide bridging. Strong support for this conclusion was obtained from electrophoretic analyses in gels containing dodecylsulfate, in that cold-insoluble globulin manifested an increased rate of migration after reduction of disulfide bridges. The reduced cold-insoluble globulin band could be resolved into a closely spaced doublet, the components of which had molecular weights of 220 000 and 215 000, respectively. Since in sedimentation equilibrium experiments the molecular weight of the unreduced molecule was estimated to be 450 000, values in this range for the size of the subunit chain suggested that each cold-insoluble globulin molecule is composed of two covalently linked chains. The nature of the size heterogeneity of the reduced subunit chains is uncertain. However, the finding of a single type of NH2-terminal sequence ([Glu-Ala) in cold-insoluble globulin preparations, is consistent with the speculation that the smaller subunit may be a catabolic intermediate arising via release of peptide material containing the COOH-terminus of a parent chain.