Glycoprotein Residues Research Articles

C0347 Thrombin binding to platelets of young patients with a history of acute myocardial infarction

The binding of alpha-thrombin (FIIa) to platelets is influenced by sulfation of three tyrosine (Tyr) residues in glycoprotein (GP) Ib alpha. Platelets of mice expressing human GPIb mutated at these residues bind 10% of the FIIa that binds to platelets expressing wild type human GPIb (hWT).

Influenza virus binds its host cell using multiple dynamic interactions

Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609-9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus-cell binding quantitatively at the molecular level.

The Hypothesis on Function of Glycosphingolipids and ABO Blood Groups Revisited

Twenty-five years ago the author proposed new ideas of glycoprotein (GPs) and glycosphingolipid (GSLs) functions at the cell membrane. The GPs, apart from their glycan carrying capacity, were assumed to have specific, protein associated, functions. In contrast, GSLs such as those of globo and neolacto/lacto series, were considered to be energetically cheap membrane packing substances, filling in membrane spaces not covered with functional GPs. The terminal carbohydrate structures of the neolacto/lacto GSLs, i.e., sialic acid residues and ABH glycotopes, were postulated to have either regulatory or protective functions, respectively. A special active role was ascribed to terminal β-galactosyl residues of GSLs and GPs. Gangliosides were considered to be functional GSLs. In the present review the author discusses these old ideas in context of the contemporary knowledge and comes to the conclusion that they have not aged.

Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer

SummaryBackgroundA universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression.MethodsIn the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy.ResultsThe majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells.ConclusionAll these results validate the importance of core-fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets.

Adhesion and biofilm formation of Mycoplasma pneumoniae on an abiotic surface

We demonstrated that when M. pneumoniae was grown on an abiotic surface of either glass or polystyrene with a serum-containing medium, the bacteria adhered to the surface and formed highly differentiated volcano-like biofilm structures. As adherence to the surface and/or biofilm formation was totally inhibited by anti-P1 polyclonal monospecific antibodies, we suggest that the adherence of M. pneumoniae to the abiotic surface and/or biofilm formation is associated with P1, the major tip organelle protein of this organism. Furthermore, adherence and/or biofilm formation was markedly inhibited by treating the serum component of the growth medium with neuraminidase or by growing the bacteria in the presence of sialyllactose, suggesting that the initial step in the adherence to and/or biofilm formation by M. pneumoniae on an abiotic surface is the interaction of the bacterium through its tip organelle with sialic acid residues of serum glycoproteins.

Two gene clusters co‐ordinate for a functional N‐acetylglucosamine catabolic pathway in Vibrio cholerae

Pathogenic microorganisms like Vibrio cholerae are capable of adapting to diverse living conditions, especially when they transit from their environmental reservoirs to human host. V. cholerae attaches to N-acetylglucosamine (GlcNAc) residues in glycoproteins and lipids present in the intestinal epithelium and chitinous surface of zoo-phytoplanktons in the aquatic environment for its survival and colonization. GlcNAc utilization thus appears to be important for the pathogen to reach sufficient titres in the intestine for producing clinical symptoms of cholera. We report here the involvement of a second cluster of genes working in combination with the classical genes of GlcNAc catabolism, suggesting the occurrence of a novel variant of the process of biochemical conversion of GlcNAc to Fructose-6-phosphate as has been described in other organisms. Colonization was severely attenuated in mutants that were incapable of utilizing GlcNAc. It was also shown that N-acetylglucosamine specific repressor (NagC) performs a dual role - while the classical GlcNAc catabolic genes are under its negative control, the genes belonging to the second cluster are positively regulated by it. Further application of tandem affinity purification to NagC revealed its interaction with a novel partner. Our results provide a genetic program that probably enables V. cholerae to successfully utilize amino - sugars and also highlights a new mode of transcriptional regulation, not described in this organism.

ChemInform Abstract: The Tn Antigen — Structural Simplicity and Biological Complexity

AbstractReview: 335 refs.

Influenza Virus Adhesion to Living Cells Measured by Single Virus Force Spectroscopy (SVFS) and Force Probe MD Simulation

Influenza virus belongs to a wide range of viruses that are enclosed in a lipid envelope. The major spike protein of the viral envelope hemagglutinin (HA) binds sialic acid (SA) residues of glycoproteins on the plasma membrane of the host cells. This represents the first step of infection and requires multiple simultaneous interactions since the affinity between one single HA-SA pair is very low (10-13 M-1).The binding interaction of influenza virus adhesion to living cells was probed by means of dynamic force spectroscopy and force probe molecular dynamics (MD) simulation. We applied three independent approaches to measure the unbinding force between influenza virus and a host cell membrane. Using optical tweezers and AFM based single molecule force spectroscopy we were able to characterize the binding energy on the single molecule level. Unbinding events where analysed and revealed a multimodal rupture force distribution which suggests sequential binding of multiple receptors. We determined the interacting force between hemagglutinin and its receptor sialic acid to be ∼10pN. Furthermore we used molecular dynamics simulation to gain information about the binding architecture and the sequence of the unbinding process. MD simulation allowed us a more detailed view of the energy landscape that governs the interaction between HA and its ligand.The combination of experimental and simulated force spectroscopy covers a very large force regime and provides information that could not be obtained with either one or the other method. The techniques are complementary and provide detailed insights of the molecular interactions involved in influenza virus attachment.

The Tn Antigen—Structural Simplicity and Biological Complexity

Glycoproteins in animal cells contain a variety of glycan structures that are added co‐ and/or posttranslationally to proteins. Of over 20 different types of sugar–amino acid linkages known, the two major types are N‐glycans (Asn‐linked) and O‐glycans (Ser/Thr‐linked). An abnormal mucin‐type O‐glycan whose expression is associated with cancer and several human disorders is the Tn antigen. It has a relatively simple structure composed of N‐acetyl‐d‐galactosamine with a glycosidic α linkage to serine/threonine residues in glycoproteins (GalNAcα1‐O‐Ser/Thr), and was one of the first glycoconjugates to be chemically synthesized. The Tn antigen is normally modified by a specific galactosyltransferase (T‐synthase) in the Golgi apparatus of cells. Expression of active T‐synthase is uniquely dependent on the molecular chaperone Cosmc, which is encoded by a gene on the X chromosome. Expression of the Tn antigen can arise as a consequence of mutations in the genes for T‐synthase or Cosmc, or genes affecting other steps of O‐glycosylation pathways. Because of the association of the Tn antigen with disease, there is much interest in the development of Tn‐based vaccines and other therapeutic approaches based on Tn expression.

N-glycosylation of zona glycoproteins during meiotic maturation is involved in sperm-zona pellucida interactions of porcine oocytes

The objective was to determine whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation of porcine oocytes, and whether this had a role in fertilization. In the first of three experiments, carbohydrate residues in the ZP of in vitro matured porcine oocytes were blocked with various lectins and the influence of such blocking on sperm-ZP interactions was studied. The second experiment used a lectin-binding assay to determine whether the number of GlcNAc residues in ZP was changed by N-glycosylation during in vitro maturation (IVM) of porcine oocytes. The last experiment determined the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM, on sperm-ZP interactions in porcine oocytes. The primary findings were that: 1) N-glycosylation of GlcNAc residues in porcine ZP occurred during the first 24 h of IVM; and 2) such glycosylation was indispensible for sperm-ZP interactions, e.g., number of sperm bound to ZP, acrosome-reacted sperm, sperm penetration rate, and level of polyspermy (P < 0.05). However, blocking N-glycosylation by tunicamycin treatment during IVM did not adversely influence the progression of oocytes to meiotic metaphase II and male pronucleus formation, indicating that this glycosylation was involved only in the initial stages of fertilization. We inferred that the increase in terminal GlcNAc residues in ZP glycoprotein through new N-glycosylation during the first 24 h of meiotic maturation played a critical role in porcine ZP acquiring the capacity to accept sperm.

Alleviation of Neurotoxicity by Microglial Human Siglec-11

Sialic acid-binding Ig superfamily lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-11 is a recently identified human-specific CD33-related Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells. Polysialylated neuronal cell adhesion molecule (PSA-NCAM) is a putative ligand of Siglec-11. We observed gene transcription and protein expression of Siglec-11 splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis. Siglec-11 was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec-11 splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells. Stimulation of Siglec-11 by cross-linking suppressed the lipopolysaccharides (LPS)-induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia. Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec-11 transduced microglia. Expression of PSA-NCAM was detected on microglia and neurons by immunohistochemistry and RT-PCR. Coculture of microglia transduced with Siglec-11 and neurons demonstrated neuroprotective function of Siglec-11. The neuroprotective effect of Siglec-11 was dependent on polysialic acid (PSA) residues on neurons, but independent on PSA on microglia. Thus, data demonstrate that human Siglec-11 ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

IL-1β and TNF-α alter the glycophenotype of primary human chondrocytes in vitro

Despite the significance of glycoproteins for extracellular matrix assembly in cartilage tissue, little is known about the regulation of the chondrocyte glycophenotype under inflammatory conditions. The present study aimed to assess the effect of IL-1β and TNF-α on specific features of the glycophenotype of primary human chondrocytes in vitro. Using LC–MS, we found that both cytokines increased overall sialylation of N- and O-glycans and induced a shift towards α-(2→3)-linked sialic acid residues in chondrocyte glycoproteins. These results were supported by quantitative PCR showing increased expression of α-(2→3) sialyltransferases in treated cells. Moreover, we found that both IL-1β and TNF-α induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans. In contrast, core α-(1→6)-fucosylation of chondrocyte N-glycans was found to be reduced particularly by TNF-α. In summary, inflammatory conditions induce specific alterations of the chondrocyte glycophenotype which might affect cell–matrix interactions or the function of endogenous lectins.

Measuring Adhesion Forces Between Influenza Virus and Living Cells

Influenza virus belongs to a wide range of viruses that are enclosed in a lipid envelope. The major spike protein of the viral envelope hemagglutinin (HA) binds sialic acid (SA) residues of glycoproteins on the plasma membrane of the host cells. This represents the first step of infection and requires multiple simultaneous interactions since the affinity between one single HA-SA pair is estimated to be very low (10-13 M-1). The attachment of influenza virus particles to living host cells was characterised on the level of single molecules using optical tweezers and atomic force spectroscopy. Unbinding events where analysed and revealed a multimodal rupture force distribution. This suggests sequential binding of multiple receptors. Treatment of the cells with neuraminidase (NA) which cleaves terminal sialic acid residues leads to a decrease of the binding probability by >50 %. This indicates a specific interaction between hemagglutinin and sialic acid unravelled by force measurements.

Inhibition of Influenza Virus Activity by Sialic Acid Conjugated Multivalent Particles

Influenza virus binds via its envelope protein hemagglutinin (HA) sialic acid (SA) residues of glycoproteins on the plasma membrane of the host cell. The affinity of a single HA-SA pair is low (103 M−1) compared to the overall affinity between virus and cell surface caused by multiple simultaneous interactions. For example, the affinity between virus and erythrocytes - serving as a model for a host cell - is about 1013 M−1. Multivalency is ubiquitous in biology and can dramatically enhance affinities.

Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase fromStreptococcus pneumoniae

Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2(1) and diffracted to 2.0 A resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 A, beta = 112.46 degrees. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-alpha-D-galactopyranoside, which is consistent with the classification of this enzyme as an endo-alpha-N-acetylgalactosaminidase.

Characterization of the gene encoding glycoprotein C of duck enteritis virus

A total of 2,718 bp of DNA fragment was amplified from the C-KCE strain of duck enteritis virus (DEV) genome using thermal asymmetric interlaced PCR. This newly identified viral DNA fragment contained two non-overlapping open reading frames (ORFs) oriented from the 5' to 3' direction. The first ORF was comprised of 43.5% G + C and contained the full-length genomic sequence of the UL44 gene (1,296 bp) encoding 431 amino acid residues of DEV glycoprotein C (gC). The second ORF encoded a partial peptide of the UL43 gene. The sequences of DNA and deduced amino acids of the DEV gC gene shared high homology with other members of known herpesviruses, supporting the classification of DEV. Phylogenetic analysis of the DEV gC gene revealed that the gC gene had a close evolutionary relationship with the subfamily of Alphaherpesvirinae.

Simple fluorimetric method for quantification of sialic acids in glycoproteins

A simple and rapid fluorimetric method was developed for detection and quantitative analysis of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0°C for 45min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine derivative with acetoacetanilide and ammonia at room temperature for 10min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471nm, respectively. When the reaction was conducted in approximately a 1-ml volume, the linearity of the calibration exhibited between 2 and 180μg of bovine fetuin, or between 0.3 and 27nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the standard deviation of the reagent blank, was 0.5μg of fetuin. The proposed method was applied to determination of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extraction steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.

Complex of a Protective Antibody with Its Ebola Virus GP Peptide Epitope: Unusual Features of a Vλ x Light Chain

13F6-1-2 is a murine monoclonal antibody that recognizes the heavily glycosylated mucin-like domain of the Ebola virus virion-attached glycoprotein (GP) and protects animals against lethal viral challenge. Here we present the crystal structure, at 2.0 Å, of 13F6-1-2 in complex with its Ebola virus GP peptide epitope. The GP peptide binds in an extended conformation, anchored primarily by interactions with the heavy chain. Two GP residues, Gln P406 and Arg P409, make extensive side-chain hydrogen bond and electrostatic interactions with the antibody and are likely critical for recognition and affinity. The 13F6-1-2 antibody utilizes a rare Vλ x light chain. The three light-chain complementarity-determining regions do not adopt canonical conformations and represent new classes of structures distinct from Vκ and other Vλ light chains. In addition, although Vλ x had been thought to confer specificity, all light-chain contacts are mediated through germ-line-encoded residues. This structure of an antibody that protects against the Ebola virus now provides a framework for humanization and development of a postexposure immunotherapeutic.

Resistance to deglycosylation by ammonia of IgA1 O-glycopeptides: implications for the β-elimination of O-glycans linked to serine and threonine

Pools of O-glycopeptides (and their deglycosylated analogues) derived from trypsin-digested normal human serum IgA1 have been treated with ammonia under conditions reported to result in complete liberation of O-glycans linked to serine and threonine residues in glycopeptides and glycoproteins. MALDI-TOF MS analysis has revealed that only one of the six glycosylated sites is susceptible to β-elimination under these conditions. It is likely that resistance to β-elimination is due to very close proximity of proline to the glycosylated serine or threonine residues. Preliminary results using 0.1 M NaOH (instead of ammonia) to perform β-elimination indicated that there was also selective de-O-glycosylation with this reagent, however, these results were complicated by the concomitant hydrolysis of the peptide bonds. These findings may have implications for similarly O-glycosylated peptides and proteins and possibly for other chemical methods that are used to carry out β-eliminations of O-glycans.

The Elastin Receptor Complex Transduces Signals through the Catalytic Activity of Its Neu-1 Subunit

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.