Role Of Glycosylation Research Articles

β-1,4-galactosyltransferase III suppresses extravillous trophoblast invasion through modifying β1-integrin glycosylation

IntroductionGlycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The β-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. MethodsB4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. ResultsB4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on β1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced β1-integrin degradation. DiscussionWe propose that B4GALT3-mediated glycosylation change not only enhances β1-integrin binding to laminin, but also attenuates β1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy.

Role of glycosylation in secretion and stability of micromycetes α-galactosidase.

The effect of the glycosylation inhibitors (tunicamycin and 2-deoxy-D-glucose) on the activity, stability and production of fungal glycosidases has been studied. It was shown that inhibition of N-glycosylation sites did not affect the secretion of Aspergillus niger α-galactosidase, however reduced yield of Cladosporium cladosporioides and Penicillium canescens α-galactosidases. Changes in the level of O-glycosylation resulted in a significant reduction in the activity and stability of α-galactosidases of all three producers tested. Activity of the modified enzymes was significantly lower than that of the native ones, and was 2.6 and 0.33 U/mg for A. niger α-galactosidase, 3.3 and 32.5 U/mg for C. cladosporioides α-galactosidase, 11.66 and 31.1 U/mg for P. canescens α-galactosidase, respectively. A. niger α-galactosidase completely lost activity during purification and storage. The decrease of thermal stability at 55 °C by 20% was shown for C. cladosporioides and P. canescens α-galactosidases. It was also noted that O-deglycosylation led to a decrease in resistance of these enzymes to the action of proteases.

Complex N‐linked glycans serve as a determinant for exosome/microvesicle cargo recruitment (788.5)

Exosomes, also known as microvesicles (EMVs), are nano‐sized membrane particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell‐cell signaling, immune activation and suppression, and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein cargo, which can dictate pathogenicity. While some mechanisms have been proposed for the regulation of EMV cargo selection, little attention has been paid to N‐linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from the overall cellular membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we explore further the role of glycosylation in EMV cargo selection. We identify specific glycosylated targets and associated proteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Further, we show that genetic manipulation of the glycosylation levels of a MV cargo glycoprotein EWI‐2 directly impacts its recruitment as a function of N‐linked glycan sites. Taken together, our data provided strong evidence that N‐linked glycosylation directs glycoprotein sorting into EMVs and serves as a determinant of EMV cargo selection.Grant Funding Source: Supported by Mizutani Foundation for Glycoscience

The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

Proteases from Russell's viper venom (RVV) induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

Diversity, Abundance and Distribution of O-linked Glycosylation Pathway Enzymes in Prokaryotes-A Comparative Genomics Study

In prokaryotes, the protein protein N- and O-glycosylation pathways (GlyPW) have been experimentally characterised in some of the organisms. Identifying GlyPWs in other prokaryotes is essential to understand the role of glycosylation. Herein we report a BLASTp and a hidden Markov model (HMM)-profile based comparative genomics approach to identify putative O-glycosylation enzymes in completely sequenced prokaryotic genomes using the experimentally characterized O-GlyPW enzymes as query sequences. Homologs for enzymes of all five categories viz., initiation, modification, extension, flippase and oligosaccharyltransferase are found in 128 organisms and no homolog is found for any of these in 52 organisms. A large number of organisms have homologs for all categories except oligosaccharyltransferases, which show high sequence diversity. Thus, O-GlyPW enzyme homologs are widely prevalent. Most of the 128 organisms are proteobacteria and more than half are pathogenic. The pattern of distribution of homologs indicates species- and strainspecific variations and acquisition of homologs by horizontal gene transfer.

Golgi‐mediated Glycosylation Determines Residency of the T2 RNase Rny1p in Saccharomyces cerevisiae

The role of glycosylation in the function of the T2 family of RNases is not well understood. In this work, we examined how glycosylation affects the progression of the T2 RNase Rny1p through the secretory pathway in Saccharomyces cerevisiae. We found that Rny1p requires entering into the ER first to become active and uses the adaptor protein Erv29p for packaging into COPII vesicles and transport to the Golgi apparatus. While inside the ER, Rny1p undergoes initial N-linked core glycosylation at four sites, N37, N70, N103 and N123. Rny1p transport to the Golgi results in the further attachment of high-glycans. Whereas modifications with glycans are dispensable for the nucleolytic activity of Rny1p, Golgi-mediated modifications are critical for its extracellular secretion. Failure of Golgi-specific glycosylation appears to direct Rny1p to the vacuole as an alternative destination and/or site of terminal degradation. These data reveal a previously unknown function of Golgi glycosylation in a T2 RNase as a sorting and secretion signal.

Enhanced Activity of Rhizomucor miehei Lipase by Deglycosylation of Its Propeptide in Pichia pastoris

Many studies have demonstrated that the properties of enzymes expressed in eukaryotes can be affected by the position and extent of glycosylation on enzyme. In this study, two potential glycosylation sites (the 8th and the 58th asparagine) were identified and the effect of propeptide glycosylation on Rhizomucor miehei lipase (RML) expressed in Pichia pastoris was investigated. To better understand the effect of glycosylation on the activity of RML, three mutants (M1, generated by N8A; M2, generated by N58A; and M3, generated by N8A and N58A) were designed to generate deglycosylated enzymes. The results showed that deglycosylated RML exhibited a twofold higher activity compared to the wild type. However, it was also found that glycosylation on the propeptide was important for the removal of the propeptide by Kex2 protease and secretion of the enzyme. Thus, our study provided a further understanding into the role of glycosylation on enzyme function.

A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21-108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics simulations were consistent with these observations. Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies.

Role of glycosylation in the anticancer activity of antibacterial peptides against breast cancer cells

Antibacterial peptides (ABPs) with cancer-selective toxicity have received much more attention as alternative chemotherapeutic agents in recent years. However, the basis of their anticancer activity remains unclear. The modification of cell surface glycosylation is a characteristic of cancer cells. The present study investigated the effect of glycosylation, in particular sialic acid, on the anticancer activity of ABPs. We showed that aurein 1.2, buforin IIb and BMAP-28m exhibited selective cytotoxicity toward MX-1 and MCF-7 breast cancer cells. The binding activity, cytotoxicity and apoptotic activity of ABPs were enhanced by the presence of O-, N-glycoproteins, gangliosides and sialic acid on the surface of breast cancer cells. Among N-, O-glycoproteins and ganglioside, O-glycoproteins almost had the strongest effect on the binding and cytotoxicity of the three peptides. Further, up-regulation of hST6Gal1 in CHO-K1 cells enhanced the susceptibility of cells to these peptides. Finally, the growth of MX-1 xenograft tumors in mice was significantly suppressed by buforin IIb treatment, which was associated with induction of apoptosis and inhibition of vascularization. These data demonstrate that the three peptides bind to breast cancer cells via an interaction with surface O-, N-glycoproteins and gangliosides. Sialic acids act as key glycan binding sites for cationic ABP binding to glycoproteins and gangliosides. Therefore, glycosylation in breast cancer cells plays an important role in the anticancer activity of ABPs, which may partly explain their cancer-selective toxicity. Anticancer ABPs with cancer-selective cytotoxicity will be promising candidates for anticancer therapy in the future.

Purification and characterization of a betanidin glucosyltransferase from Amaranthus tricolor L catalyzing non-specific biotransformation of flavonoids

Betacyanins are the major pigments present in Amaranthus tricolor, a leafy vegetable consumed globally. The terminal glycosyltaion of the aglycone betanidin is an important step in the biosynthesis of this natural red antioxidant pigment. A betanidin 5-O-glucosyltransferase (BGT) was fully purified to 134 folds (specific activity, 265.2nkatmg−1) from the red amaranth by ammonium sulfate precipitation followed by hydrophobic interaction, anion exchange and size exclusion chromatography. Homogeneity of the purified protein was confirmed by 2-dimensional polyacrylamide gel electrophoresis (2D PAGE). The molecular weight of the enzyme determined by liquid chromatography–mass spectrometry (LC–MS) was found to be 62.8kDa. Furthermore, the enzyme glycosylated flavonoids (kaempferol and quercetin) but not anthocyanidins, presence of which is mutually exclusive to betacyanin accumulating plants. The apparent Km (344±2.34μM) and Vmax (17.24μMmin−1) of the enzyme were determined by LC–MS/MS. Peptide mass fingerprinting of the purified protein showed 38.4% coverage of peptide masses with anthocyanidin 3-O-glucosyltransferase from Zea mays. Study on this purified enzyme, for the first time, revealed its role of glycosylation in biosynthesis of betacyanin in A. tricolor and indicates promiscuous substrate-specificity.

Mummy, A UDP-N-acetylglucosamine pyrophosphorylase, modulates DPP signaling in the embryonic epidermis of Drosophila

The evolutionarily conserved JNK/AP-1 (Jun N-terminal kinase/activator protein 1) and BMP (Bone Morphogenetic Protein) signaling cascades are deployed hierarchically to regulate dorsal closure in the fruit fly Drosophila melanogaster. In this developmental context, the JNK/AP-1 signaling cascade transcriptionally activates BMP signaling in leading edge epidermal cells. Here we show that the mummy (mmy) gene product, which is required for dorsal closure, functions as a BMP signaling antagonist. Genetic and biochemical tests of Mmy's role as a BMP-antagonist indicate that its function is independent of AP-1, the transcriptional trigger of BMP signal transduction in leading edge cells. pMAD (phosphorylated Mothers Against Dpp) activity data show the mmy gene product to be a new type of epidermal BMP regulator – one which transforms a BMP ligand from a long- to a short-range signal. mmy codes for the single UDP-N-acetylglucosamine pyrophosphorylase in Drosophila, and its requirement for attenuating epidermal BMP signaling during dorsal closure points to a new role for glycosylation in defining a highly restricted BMP activity field in the fly. These findings add a new dimension to our understanding of mechanisms modulating the BMP signaling gradient.

The Polysialyltransferases Interact with Sequences in Two Domains of the Neural Cell Adhesion Molecule to Allow Its Polysialylation

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an "extra" N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.

Evaluation of Molecular Species of Prostate-Specific Antigen Complexed with Immunoglobulin M in Prostate Cancer and Benign Prostatic Hyperplasia

This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulin M (IgM). Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.

Is High Throughput Glycomics Possible?

The role of glycosylation and their biological functions whether as free oligosaccharides or glycoconjugates has been made possible by the recent advancements in the analyses of these compounds. The heterogeneity and the large structural diversity have made oligosaccharide analysis significantly more difficult than other biopolymers. The next stage of development is to achieve high throughput analysis. However, the structural elucidation of oligosaccharides remains an extremely difficult task. Recent reports reveal that the diversity of structures in a given biological system is finite and may not be large. It may be possible to create a database of structures that can be used to determine the identity of known compounds. This capability would therefore make high throughput glycomics possible. Achieving this task depends on the proper selection of chemical characteristics to identify the compound. In this presentation, nanoflow liquid chromatography retention times, accurate mass, and tandem MS is used to determine structure with a high degree of certainty. The method is used to determine the biological function of milk oligosaccharides as well as to discover glycan-based biomarkers for diseases.

Differential immunogenicity and allergenicity of native and recombinant human lactoferrins: Role of glycosylation

Human native milk lactoferrin (LF) and recombinant forms of lactoferrin (rLF) are available with identical aa sequences, but different glycosylation patterns. Native lactoferrin (NLF) possesses the intrinsic ability to stimulate vigorous IgG and IgE antibody responses in BALB/c mice, whereas recombinant forms (Aspergillus or rice) are 40-fold less immunogenic and 200-fold less allergenic. Such differences are independent of endotoxin or iron content and the glycans do not contribute to epitope formation. A complex glycoprofile is observed for NLF, including sialic acid, fucose, mannose, and Lewis (Le)(x) structures, whereas both rLF species display a simpler glycoprofile rich in mannose. Although Le(x) type sugars play a Th2-type adjuvant role, endogenous expression of Le(x) on NLF did not completely account for the more vigorous IgE responses it provoked. Furthermore, coadminstration of rLF downregulated IgE and upregulated IgG2a antibody responses provoked by NLF, but was without effect on responses to unrelated peanut and chicken egg allergens. These results suggest glycans on rLF impact the induction phase to selectively inhibit IgE responses and that differential glycosylation patterns may impact on antigen uptake, processing and/or presentation, and the balance between Th1 and Th2 responses.

GOLGA2/GM130, cis-Golgi Matrix Protein, is a Novel Target of Anticancer Gene Therapy

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

UDP-N-acetylglucosamine transporter and UDP-galactose transporter form heterologous complexes in the Golgi membrane

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) are evolutionarily related. We hypothesize that their role in glycosylation may be coupled through heterologous complex formation. Coimmunoprecipitation analysis and FLIM–FRET measurements performed on living cells showed that NGT and UGT form complexes when overexpressed in MDCK-RCAr cells. We also postulate that the interaction of NGT and UGT may explain the dual localization of UGT2. For the first time we demonstrated in vivo homodimerization of the NGT nucleotide sugar transporter. In conclusion, we suggest that NGT and UGT function in glycosylation is combined via their mutual interaction. Structured summary of protein interactionsNGTphysically interacts with UGT2 by anti tag coimmunoprecipitation (View Interaction: 1, 2)NGTphysically interacts with UGT1 by anti tag coimmunoprecipitation (View interaction)UGT2physically interacts with NGT by fluorescent resonance energy transfer (View interaction)NGTphysically interacts with NGT by fluorescent resonance energy transfer (View interaction)UGT1physically interacts with UGT2 by anti tag coimmunoprecipitation (View interaction)UGT1physically interacts with NGT by fluorescent resonance energy transfer (View interaction)

Putative Glycosyltransferases and Other Plant Golgi Apparatus Proteins Are Revealed by LOPIT Proteomics

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

P144 Pathogen recognition by the long pentraxin PTX3: Roles of glycosylation and interplay with complement

Introduction The long pentraxin PTX3 is a soluble Pattern Recognition Molecule (sPRM) with non-redundant roles in resistance to selected pathogens, such as the opportunistic fungus Aspergillus fumigatus . PTX3 facilitates recognition and phagocytosis of fungal conidia mainly by polymorphonuclear neutrophils (PMNs) via Fc γ receptors (Fc γ Rs) and alternative complement pathway (ACP) mechanisms. The human PTX3 protein contains a single N-glycosylation site with complex type sialylated oligosaccharides that has been involved in a number of PTX3 functions [1] . The present study was designed to characterize the role of this post translational modification in the PTX3-depedent recognition and phagocytosis of A. fumigatus . This microorganism has developed several strategies to evade the immune response, such as attracting onto the conidial wall inhibitors of the ACP, like factor H (FH, a ligand of PTX3). Here we present preliminary evidences on the effect of PTX3 on FH engagement by A. fumigatus . Methods Recombinant as well as natural human PTX3 (i.e., from PMNs) was purified by immunoaffinity chromatography and the attached oligosaccharides were analyzed using glycosidases, lectin blotting and mass spectrometry. A range of PTX3 glycoforms were made in vitro by enzymatic hydrolysis of terminal monosaccharides of the recombinant protein. The opsonic and pro-phagocytic activities of these variants were characterized in buffer solutions and whole human peripheral blood, respectively, by flow cytometry. The influence of PTX3 on FH binding to conidia was studied in competition assays using microtiter plates. Results Complex-type sugars were found on PTX3 of both natural and recombinant origins. These showed heterogeneity with regard to the relative content of terminal sialic acid (SA) and bi-, tri- and tetrantennary glycans, depending on the cellular source. Enzymatic removal of SA caused ∼50% reduction of PTX3 binding to A. fumigatus conidia, with no further change due to additional deglycosylation. Conversely, saccharide hydrolysis had no effect on PTX3 binding to PMNs, where Fc γ Rs have been proposed as putative receptors of this pentraxin [1] . However, consistent with binding data from conidia, desialylated PTX3 showed reduced potentiation of conidia phagocytosis by PMNs as compared to the unmodified protein. Also, we observed that PTX3 inhibits the binding of FH to A. fumigatus conidia, even when these were pre-opsonised with the long pentraxin and FH was applied at physiologically relevant concentrations. Conclusion Different pathogens express neuraminidases on their surface during infection and natural immunity cells, such as PMNs, mobilize intracellular neuraminidases to the plasma membrane upon inflammation. Here, we report that the glycosylation status of the sPRM PTX3 changes, with respect to SA, depending on cellular source and perhaps inducing stimuli. We describe that SA modulates the PTX3-dependent recognition and phagocytosis of A. fumigatus , which points to glycosylation as a strategy to tune PTX3 functions in innate immunity. Furthermore, we provided evidences that PTX3 impairs the interaction of FH with A. fumigatus , where this might link to a new mechanism to counterbalance the complement evasion strategy exploited by this pathogen.

Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

BackgroundEnterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear.ResultsWe demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA) and Sambucus Nigra (SNA), could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation.ConclusionsIn this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.