Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV.

Sialic acid-binding immunoglobulin-like lectins (siglecs) are predominately expressed on immune cells. They are best known as regulators of cell signaling mediated by cytoplasmic tyrosine motifs and are increasingly recognized as receptors for pathogens that bear sialic acid-containing glycans. Most siglec proteins undergo endocytosis, an activity tied to their roles in cell signaling and innate immunity. Here, we investigate the endocytic pathways of two siglec proteins, CD22 (Siglec-2), a regulator of B-cell signaling, and mouse eosinophil Siglec-F, a member of the rapidly evolving CD33-related siglec subfamily that are expressed on cells of the innate immune system. CD22 exhibits hallmarks of clathrin-mediated endocytosis and traffics to recycling compartments, consistent with previous reports demonstrating its localization to clathrin domains. Like CD22, Siglec-F mediates endocytosis of anti-Siglec-F and sialoside ligands, a function requiring intact tyrosine-based motifs. In contrast, however, we find that Siglec-F endocytosis is clathrin and dynamin independent, requires ADP ribosylation factor 6, and traffics to lysosomes. The results suggest that these two siglec proteins have evolved distinct endocytic mechanisms consistent with roles in cell signaling and innate immunity.

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet circulation lifetime is not fully clarified. We show that thrombocytopenia in mice deficient in the St3gal4 sialyltransferase gene (St3Gal-IV(-/-) mice) is caused by the recognition of terminal galactose residues exposed on the platelet surface in the absence of sialylation. This results in accelerated platelet clearance by asialoglycoprotein receptor-expressing scavenger cells, a mechanism that was recently shown to induce thrombocytopenia during Streptococcus pneumoniae sepsis. We now identify platelet GPIbalpha as a major counterreceptor on ST3Gal-IV(-/-) platelets for asialoglycoprotein receptors. Moreover, we report data that establish the importance of sialylation of the von Willebrand factor in its function.

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ~20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an "expanded" rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.

BACKGROUND

Obesity and obesity-associated type 2 diabetes mellitus (T2DM) are frequently related to a low-grade chronic inflammatory state, which increases the risk of developing cardiovascular diseases. The aim of the present work was to evaluate the effect of obesity and T2DM on the concentrations of pro-inflammatory factors and to study the effect of weight loss after Roux-en-Y gastric bypass (RYGBP).

METHODS

Plasma concentrations of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-alpha), serum amyloid A (SAA) and sialic acid (SA) were measured in 25 female volunteers. The concentrations of these cytokines were determined in 14 female obese patients before and after weight loss following RYGBP. Additionally, visceral adipose tissue (VAT) obtained from 15 females was used to quantify expression levels of MCP-1 and CD68 by Real-Time PCR.

RESULTS

Both obese normoglycemic (NG) and T2DM groups exhibited significantly higher MCP-1 (P < 0.05), TNF-alpha (P < 0.01), SAA (P < 0.05) and SA (P < 0.05) concentrations, compared to the lean group. No differences were found between obese NG and obese T2DM subjects. A significant positive correlation was found between body fat percentage (BF) and all inflammatory markers (P < 0.05) studied. MCP-1 expression levels in VAT were upregulated in obese NG (P = 0.008) and obese T2DM (P = 0.032) patients compared to lean subjects, but no additional detrimental effect of T2DM was observed between both obese groups. After weight loss, SAA (P < 0.001) and SA (P < 0.05) concentrations diminished, whereas circulating levels of MCP-1 showed a tendency to decrease (P = 0.093) and TNF-alpha did not change.

CONCLUSIONS

The present findings suggest that elevated pro-inflammatory cytokine levels found in obese patients relate mainly to obesity rather than to T2DM. Moreover, surgery-induced weight loss reduces circulating concentrations of key pro-inflammatory factors, which contribute to the improvement in the cardiovascular co-morbidity following excess weight loss.

Integrins alpha2beta1, alphaXbeta2, and alphaVbeta3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the alpha2beta1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the alphaXbeta2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of alpha2beta1, alphaXbeta2, and alphaVbeta3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound alpha2beta1, and VP7 interacted with alphaXbeta2 and alphaVbeta3 at a postbinding stage. DGEA inhibited rotavirus binding to alpha2beta1 and infectivity, whereas GPRP binding to alphaXbeta2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed alpha2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the alpha2 I domain. In a novel process, integrin-using viruses bind the alpha2 I domain of alpha2beta1 via DGE in VP4 and interact with alphaXbeta2 (via GPR) and alphaVbeta3 by using VP7 to facilitate cell entry and infection.

Antibodies to the alpha and beta 2 subunits and site-directed antibodies that distinguish alpha subunits of the RI and RII subtypes have been used to study the biosynthesis and assembly of sodium channels. The RII sodium channel subtype is preferentially expressed in rat brain neurons in primary cell culture. Post-translational processing of alpha subunits includes incorporation of palmityl residues in thioester linkage and sulfate residues attached to oligosaccharides. The incorporation of [3H] palmitate into alpha subunits is inhibited by tunicamycin, indicating that it occurs in the early stages of biosynthesis but after co-translational glycosylation. Mature alpha subunits are attached to beta 2 subunits through disulfide bonds within 1 h after synthesis and up to 30% can be specifically immunoprecipitated from the cell surface with antibodies against the beta 2 subunits by 4 h after synthesis. The remaining alpha subunits remain in an intracellular pool. The alpha subunits synthesized in the presence of castanospermine and swainsonine have reduced apparent size. Castanospermine prevents incorporation of approximately 81% of the sialic acid of the alpha subunit and inhibits sulfation but not palmitylation. Although inhibition of glycosylation with tunicamycin blocks assembly of functional sodium channels, castanospermine and swainsonine do not prevent the covalent assembly of alpha and beta 2 subunits or the transport of alpha beta 2 complexes to the cell surface, and sodium channels synthesized under these conditions have normal affinity for saxitoxin. Thus, the extensive processing and terminal sialylation of oligosaccharide chains during maturation of the alpha subunit is not essential. A kinetic model for biosynthesis, processing, and assembly of sodium channel subunits is presented.

The complicated interplay between cancer and the host immune system has been studied for decades. New insights into the human immune system as well as the mechanisms by which tumours evade immune control have led to the new and innovative therapeutic strategies that are considered amongst the medical breakthroughs of the last few years. Here, we will review the current understanding of cancer immunology in general, including immune surveillance and immunoediting, with a detailed look at immune cells (T cells, B cells, natural killer cells, macrophages and dendritic cells), immune checkpoints and regulators, sialic acid-binding immunoglobulin-like lectins (Siglecs) and other mechanisms. We will also present examples of new immune therapies able to reverse immune evasion strategies of tumour cells. Finally, we will focus on therapies that are already used in daily oncological practice such as the blockade of immune checkpoints cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death-1 (PD-1) in patients with metastatic melanoma or advanced lung cancer, or therapies currently being tested in clinical trials such as adoptive T-cell transfer.

During the evolution of humans, an inactivating deletion was introduced in the CMAH (cytidine monophosphate-sialic acid hydroxylase) gene, which eliminated biosynthesis of the common mammalian sialic acid N-glycolylneuraminic acid from all human cells. We found that this human-specific change in sialylation capacity contributes to the marked discrepancy in phenotype between the mdx mouse model for Duchenne muscular dystrophy (DMD) and the human disease. When compared to human patients with DMD, mdx mice show reduced severity or slower development of clinically relevant disease phenotypes, despite lacking dystrophin protein in almost all muscle cells. This is especially true for the loss of ambulation, cardiac and respiratory muscle weakness, and decreased life span, all of which are major phenotypes contributing to DMD morbidity and mortality. These phenotypes occur at an earlier age or to a greater degree in mdx mice that also carry a human-like mutation in the mouse Cmah gene, possibly as a result of reduced strength and expression of the dystrophin-associated glycoprotein complex and increased activation of complement. Cmah-deficient mdx mice are a small-animal model for DMD that better approximates the human glycome and its contributions to muscular dystrophy.

Phase variation of Neisseria gonorrhoeae lipopolysaccharide (LPS) controls both bacterial entry into human mucosal cells, and bacterial susceptibility to killing by antibodies and complement. The basis for this function is a differential sialylation of the variable oligosaccharide moiety of the LPS. LPS variants that incorporate low amounts of sialic acid enter human mucosal epithelial cells very efficiently, but are susceptible to complement-mediated killing. Phase transition to a highly sialylated LPS phenotype results in equally adhesive but entry deficient bacteria which, however, resist killing by antibodies and complement because of dysfunctional complement activation. Phase variation of N. gonorrhoeae LPS thus functions as an adaptive mechanism enabling bacterial translocation across the mucosal barrier, and, at a later stage of infection, escape from the host immune defence.

Immunoglobulin M (IgM)-secreting murine plasmablasts have been used to explore the cytologic site(s) of the successive modifications of the polypeptide H and L chains (steps of glycosylation, chain assembly, and polymerization) which occur during intracellular transport (ICT) and the interrelationships between these events. A combination of pulse-chase biosynthetic labeling protocols (using amino acids and sugars), subcellular fractionation, and electron microscope autoradiography was used in conjunction with inhibitors of glycosylation and agents (carboxyl cyanide m-chlorophenyl hydrazone [CCCP] and monensin) which block Ig exit from the rough endoplasmic reticulum (RER) or Golgi cisternae. The data are consistent with the following conclusions: (1) Sugar addition and modification occur in three main steps: (a) en bloc addition of core sugars to nascent H chains, (b) partial trimming of these oligosaccharide chains in the RER, (c) quasiconcerted addition of terminal sugars (galactose, fucose, and sialic acid) in a very distal compartment between monensin-sensitive Golgi cisternae and the cell surface. (2) H and L chain assembly occurs between nascent H chains and a pool of free light chains present in the RER, followed by interchain disulfide bonding and rapid assembly of monomers into J chain-containing pentamers in the RER. Small amounts of various apparently non-obligatory intermediates in polymerization are also formed. (3) Carbohydrate addition is not required for chain assembly, polymerization, and secretion since completely unglycosylated chains (synthesized in the presence of deoxyglucose or tunicamycin) undergo polymerization and are secreted (although at a reduced rate). (4) Surface 8s IgM molecules do not represent a step in the IgM secretory pathway.

Sialidases (EC 3.2.1.18 or neuraminidases) remove sialic acid from sialoglycoconjugates, are widely distributed in nature, and have been implicated in the pathogenesis of many diseases. The three-dimensional structure of influenza virus sialidase is known, and we now report the three-dimensional structure of a bacterial sialidase, from Salmonella typhimurium LT2, at 2.0-A resolution and the structure of its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid at 2.2-A resolution. The viral enzyme is a tetramer; the bacterial enzyme, a monomer. Although the monomers are of similar size (approximately 380 residues), the sequence similarity is low (approximately 15%). The viral enzyme contains at least eight disulfide bridges, conserved in all strains, and binds Ca2+, which enhances activity; the bacterial enzyme contains one disulfide and does not bind Ca2+. Comparison of the two structures shows a remarkable similarity both in the general fold and in the spatial arrangement of the catalytic residues. However, an rms fit of 3.1 A between 264 C alpha atoms of the S. typhimurium enzyme and those from an influenza A virus reflects some major differences in the fold. In common with the viral enzyme, the bacterial enzyme active site consists of an arginine triad, a hydrophobic pocket, and a key tyrosine and glutamic acid, but differences in the interactions with the O4 and glycerol groups of the inhibitor reflect differing kinetics and substrate preferences of the two enzymes. The repeating "Asp-box" motifs observed among the nonviral sialidase sequences occur at topologically equivalent positions on the outside of the structure. Implications of the structure for the catalytic mechanism, evolution, and secretion of the enzyme are discussed.

The carbohydrate moieties of the G glycoprotein of vesicular stomatitis virus (VSV) grown in three distinct lectin-resistant (LecR) Chinese hamster ovary (CHO) cell lines have been compared by fine structural analysis of radiolabeled glycopeptides. The mutant WgaRIII, selected for resistance to wheat germ agglutinin (WGA), produces VSV containing G glycoprotein specifically lacking in sialic acid. The mutant PhaRI, selected for resistance to phytohemagglutinin (PHA) and previously shown to lack a particular glycoprotein N-acetyl-glucosaminyl-transferase activity, produces VSV containing G glycoprotein specifically lacking terminal N-acetylglucosamine-galactose-sialic acid sequences and possessing an increased number of mannose residues in the "core" region of its carbohydrate moieties. The mutant PhaRIConARII, a "double" mutant selected from PhaRI cells for resistance to concanavalin A (ConA), produces VSV containing G glycoprotein with a further alteration in the mannose residues of the "core" oligosaccharide region. We discuss the relevance of these findings to the mechanisms of glycoprotein biosynthesis in mammalian cells and to the biochemical bases of lectin resistance in CHO cells.

Distribution of lectin-binding sites in adult and developing mouse kidney was studied with fluorochrome- and peroxidase-coupled lectins. Effects of fixation methods on lectin-binding patterns were also compared. Un-induced mesenchymal cells and ureter bud of the early metanephros reacted with Concanavalin A, Lens culinaris, Ricinus communis I, and wheat germ agglutinins, whereas binding sites for both soybean and peanut (PNA) agglutinins were seen only in ureter bud tissue. On induction, PNA positivity rapidly appeared in the induced, condensed areas of the metanephrogenic mesenchyme. Early glomeruli expressed heterogeneously terminal galactosyl and N-acetylgalactosaminyl moieties in the podocytes. Later, these sites disappeared and were apparently covered by sialic acids. Endothelia also displayed a comparable sialylation of terminal saccharide moieties during maturation. Binding sites for many of the above lectins were also found in the developing proximal and distal tubules. Terminal fucosyl residues, characteristic of mature proximal tubules, appeared during day 13 of development. Dolichos biflorus agglutinin reactivity, typically seen in the collecting ducts, appeared by day 13. Griffonia simplicifolia-I-B4 isolectin reactivity was exclusively localized to endothelial in adult kidney cortex, but in embryonic kidneys reactivity with collecting duct and podocytes was also seen. These results suggest that the compartmentalized expression of cell glycoconjugates in adult mouse kidney is acquired in a sequential manner during development. Such sequential appearance of the mature glycosylation pattern probably reflects functional maturation of the nephron.

The cecal flora of mice is able to eliminate Clostridium difficile from the mouse cecum even when C. difficile is the first organism established. We used a continuous-flow (CF) culture model of the cecal flora to investigate the possibility that competition for nutrients is one mechanism for this antagonism. The medium for the CF cultures consisted of homogenates of fecal pellets from germfree mice. Carbohydrate analysis showed that mouse flora depleted 74 to 99.8% of the various carbohydrates from this environment-simulating medium. When inoculated into filtrates made from CF cultures of mouse flora, C. difficile multiplied slower than the dilution rate of the CF cultures unless glucose, N-acetylglucosamine, or N-acetylneuraminic acid was added. C. difficile did not synthesize hydrolytic enzymes able to cleave these monosaccharides from oligosaccharide side chains. As found previously, veal infusion broth did not support the growth of a microflora that could be transferred to gnotobiotic mice and fully suppress C. difficile. When mucin or monosaccharides found in mucin were added to veal infusion broth, the flora functioned normally in this regard. These data suggest that as yet unidentified organisms compete more efficiently than C. difficile for monomeric glucose, N-acetylglucosamine, and sialic acids found in colonic contents.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.

We describe the characterization of siglec-8, a novel sialic acid-binding immunoglobulin-like lectin that is expressed specifically by eosinophils. A full-length cDNA encoding siglec-8 was isolated from a human eosinophil cDNA library. Siglec-8 is predicted to contain three extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail of 47 amino acids. The siglec-8 gene mapped on chromosome 19q13.33-41, closely linked to genes encoding CD33 (siglec-3), siglec-5, siglec-6, and siglec-7. When siglec-8 was expressed on COS cells or as a recombinant protein fused to the Fc region of human IgG(1), it was able to mediate sialic acid-dependent binding to human erythrocytes and to soluble sialoglycoconjugates. Using specific monoclonal antibodies, siglec-8 could be detected only on eosinophils and hence appears to be the first example of an eosinophil-specific transmembrane receptor.

A soluble lectin which agglutinates trypsin-treated rabbit erythrocytes was purified from calf heart using affinity chromatography on asialofetuin-Sepharose. Its molecular weight was determined by gel filtration to be approximately 17,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the predominant molecular species had a molecular weight of 9,000, suggesting that the lectin is a dimer. Binding studies performed with iodinated lectin revealed that neuraminidase-treated calf erythrocytes contained approximately 5 X 10(6) lectin binding sites per cell. Native calf and rabbit erythrocytes bound the lectin, but human and rat erythrocytes required neuraminidase and trypsin treatment, respectively, for lectin binding to occur. A number of saccharides, glycopeptides, and glycoproteins possess haptene inhibitory activity toward lectin binding to erythrocytes. The most potent of these have either galactose beta leads to galactose beta leads to, galactose beta N-acetylglucosamine beta leads to, or galactose beta leads to N-acetylglucosamine beta leads to sequences at their nonreducing termini. Lactose and galactose beta 1 leads to 3N-acetylgalactosamine are the next best haptenes. Finally, alpha-linked galactose residues and free galactose are very weak haptenes. The presences of a terminal sialic acid residue impairs haptene activity in all instances. Calf heart also contains a membrane-associated lectin which is very similar but not identical with the soluble lectin. A soluble beta-galactoside binding lectin was also isolated from calf lung. It has the same molecular size and subunit structure as the soluble heart lectin and is antigenically identical. In binding studies, the pattern of inhibition by various haptenes was the same for all three lectins.

Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, alpha(v) integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.

We have previously shown that 35- and 50-kDa glycoconjugates of cultured metacyclic trypomastigotes participate in the attachment of parasites to mammalian cells. Here we show that when metacyclic trypomastigotes are incubated with [3H]sialyllactose, most of the sialic acid is transferred to these 35/50-kDa molecules in a reaction catalyzed by a parasite transsialidase. The sialic acid is incorporated in oligosaccharides of about 10 glucose units in size that are released from the glycoconjugate by mild alkaline hydrolysis. Compositional analysis reveals that the 35/50-kDa molecules are highly glycosylated proteins rich in threonine, galactose, N-acetyl-glucosamine and sialic acid. These glycoproteins can be labeled in vivo with [3H]palmitate, and the labeled fatty acid is released by glycosylphosphatidylinositol specific phospholipases C. This result, associated with the fact that they contain mannose, ethanolamine, myo-inositol, and lipid, indicate that these glycoproteins are anchored to the membrane by glycosylphosphatidylinositol. During cell invasion, these molecules appear to be capped and locally released by the parasite.

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (approximately 70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Man alpha 1-2Man alpha 1-2Man alpha 1- 6Man alpha 1-4GlcN. The glycans are substituted at the GlcN residue and at the third alpha Man distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by beta-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gonçalves, L. P. B., Wait, R., Travassos, L. R., and Mendoça-Previato, L. (1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.

CD33 is a member of the Ig superfamily that is restricted to cells of the myelomonocytic lineage but whose functions and binding properties are unknown. It shares sequence similarity with sialoadhesin, CD22, and the myelin-associated glycoprotein, which constitute the Sialoadhesin family of sialic acid-dependent cell adhesion molecules. In the present study, we show that CD33 is a fourth member of this family. As a model for sialic acid-dependent binding, human erythrocytes were derivatized with N-acetylneuraminic acid (NeuAc) in different linkages. A recombinant soluble form of CD33, Fc-CD33, bound red blood cells with a specificity similar to that of sialoadhesin, preferring NeuAc alpha 2,3Gal in N- and O-glycans over NeuAc alpha 2,6Gal in N-glycans. Fc-CD33 also bound selectively to the myeloid cell lines HL-60 and U937. However, CD33 was unable to mediate cell binding after transient expression in COS cells, despite high levels of surface expression. Pretreatment of the CD33-transfected cells with sialidase rendered them capable of mediating sialic acid-dependent binding. These results show that CD33 can function as a sialic acid-dependent cell adhesion molecule and that binding can be modulated by endogenous sialoglycoconjugates when CD33 is expressed in a plasma membrane.

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus.