N-acetylglucosaminyl Residues Research Articles

Platelet Features from Immune Thrombocytopenia Patients Refractory to Therapeutic Treatments

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder in which patients have wide variations in the number, activity and characteristics of their platelets. The goal of treatment of ITP is to raise platelet count to a level that will decrease or stop bleeding. Depending on the platelet count and the bleeding symptoms of each patient, therapeutic management is different. Pharmacological approaches employed for ITP treatment mainly aim to boost platelet count or to suppress immune system. Nevertheless, their single or combined application has been tried with a limited outcome in some ITP patients. These refractory patients constitute a clinical challenge. Identifying features of platelets from refractory ITP patients might be useful for a better understanding and management of this special clinical situation.Objective: This work aimed to determine whether platelets from refractory ITP patients have some distinguishing characteristic different from those from ITP patients responders to treatments (corticoids and thrombopoietin receptor-agonists).Methods: Citrated blood samples from 71 patients with primary chronic ITP responders to first or second line treatments and from 10 refractory ITP patients were included.Platelet surface receptors were analysed by flow cytometry (FCM, FACScan, BD Biosciences) with specific monoclonal antibodies against CD41/αIIb and CD61/β3, CD42a and CD42b.Platelet activation was assessed by FCM through PAC1-FITC binding (a mAb that recognizes activated conformation of fibrinogen receptor) after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and with 10 µM ADP.Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of caspases -3, -8, and -9 activities (BD Millipore).Platelet surface exposure of lectins were analyzed, respectively, with 1 µg/ml FITC-conjugated Wheat germ agglutinin lectin (WGA) or 1 µg/ml FITC-conjugated Erythrina cristagalli lectin (ECL). WGA binds to sialic acid and N-acetylglucosaminyl residues and ECL is a galactose specific legume lectin.Comparisons of quantitative variables were made with SPSS.22 software.Results: Platelets from refractory ITP patients showed a lower ability for being activated by TRAP or ADP agonists than those from ITP patients responders to treatments. Diminished responses to activation might be due to the reduced surface expression of fibrinogen receptor observed in platelets from refractory ITP patients (TABLE 1).Platelets from refractory ITP patients expressed more PS than those from responders-ITP patients under basal conditions. Moreover, platelets from refractory ITP patients showed higher activity of their caspases -3, -8 and -9 (TABLE 1).Platelets are known to contain sialic acid on glycoprotein carbohydrate side-chains. Abnormal activation of diverse sialidases causes a loss of sialic acid bound to the carbohydrate side chains of glycoproteins from platelets, leading to increased exposure of β-galactose which favors platelet capture through Ashwell-Morel receptors. This fact provides insights into the potential role of the analysis of the binding of lectins to glycoproteins of platelets from ITP patients.No differences were observed between groups for binding of ECL (responders ITP patients: 26.71±14.66, refractory ITP patients: 43.70 ± 26.16). In contrast, binding of WGA was significantly higher in the group of patients with refractory ITP (FIGURE 1).Conclusion: Platelets of refractory ITP patients had less capacity of activation by agonist stimulation and an enhanced apoptosis than platelets from ITP patients responders to treatments. Moreover, adhesion receptors expressed in their platelet surface were reduced and their glycosylation pattern seemed to differ from those of ITP patients responders to treatments. This observation poses the need of studying glycosylation processes and their consequence in platelet function and half-life.Work supported by grant from FIS-FEDER PI15/01457. NVB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

Platelet and Immune Characteristics of Patients with Immune Thrombocytopaenia Non Responders to Therapeutic Treatments

Introduction: Mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) appear to be multifactorial: autoantibodies, autoreactive CD8+ cytotoxic T cells, enhanced apoptosis and loss of sialic acid which mediates platelet clearance through the Ashwell-Morell receptors present in hepatocytes. Differential involvement of each of them might condition the ability of patients with ITP to respond to treatments. We aimed to examine platelet features and the immunological state of patients with ITP who do not respond to any treatment to detect the unique characteristics of this group. Methods: This was an observational, prospective and transversal study. Patients with chronic primary ITP were included: 28 ITP patients without treatment for at least 6 months (UT-ITP); 36 responders to agonists of thrombopoietin receptors (TPO-RA); and 14 ITP patients who did not respond to first- and second-line treatments (NR-ITP). A healthy control group (n=104) was also included in the study. Active caspase-3, -7, -8 or -9 were determined by flow cytometry using CaspaTag kits (Millipore, Madrid, Spain) in PRP diluted with HEPES-buffer containing 2 mM Ca2+ and 2 mM Gly-Pro-Arg-Pro (Sigma-Aldrich, Madrid, Spain) to prevent fibrin formation . Platelet surface glycan exposure was analysed by determining the binding of lectins by flow cytometry. To do so, washed platelets were incubated with 1 μg/ml Alexa fluor 488-conjugated wheat germ agglutinin lectin (WGA, Invitrogen, Spain) or with 1 μg/ml FITC-conjugated Ricinus communis agglutinin (RCA, Vector Labs, UK). WGA binds to sialic acid and N-acetylglucosaminyl residues, and RCA is a galactose-specific legume lectin which binding serves as an indirect measurement of the loss of sialic acid. Peripheral blood mononuclear cells (PBMCs) subsets were analysed by flow cytometry using specific antibodies. Experimental data was analysed using SPSS 9.0 software (SPSS Inc., Chicago, IL). Results: Platelets from TPO-RA treated and from NR-ITP patients had increased caspase-3, -7, -8 and -9 activities (Figure 1A). Platelets from NR-ITP patients exposed less sialic acid and more N-acetylglucosaminyl residues than the other groups (Figure 1B). Binding of WGA and RCA correlated with caspase activities (Table 1). Distribution of lymphocytes, monocytes and natural killer cells is shown in Table 1. NR-ITP patients had an increased proportion of B lymphocyte (LB), maybe due to a significant rise in the fraction of naive LB cells, and a diminution in LTreg subset. Whereas classical monocytes was increased, nonclassical monocyte fraction was decreased in the UT-ITP and NR-ITP groups. NR-ITP patients also presented an increased CD16+CD56bright cells fraction and a diminished NK CD16+CD56dim subset. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls (monocyte and NK cells subset distribution and LTreg count similar to control group). It is of interest to note the relationship between loss of sialic acid from platelet surface glycans and Tregs count: the most reduced surface exposure of sialic acid, the less Treg count (Figure 2). Conclusions: Platelets from NR-ITP patients had more signs of apoptosis and a different composition of surface glycans, accompanied by a diminished LTreg population, a higher LB naïve percentage, and an increased CD16+CD56bright cells fraction in circulation, indicating a severe deregulation of the immune system. Since an inverse correlation was observed between loss of sialic acid and LTreg count, a potential relationship between glycan composition on the platelet surface and immune response is suggested, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP. On the other hand, TPO-RA appears to have a beneficial effect on immune response. Nevertheless, one of the limitations of our study was that patients were recruited once the response to TPO-RA was achieved; therefore, a longitudinal study would provide more information regarding TPO-RA effects. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Canales:Novartis: Honoraria; Takeda: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Celgene: Honoraria; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Janssen: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

Platelet Protein Glycosylation in Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet count (≤100x109/L) due to platelet destruction and insufficient platelet production. There is a wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments.It has been proposed that certain autoimmune diseases might be caused by changes in glycosylation of cell surface proteins which induce the immune system to recognize cells as “non-self”. The aim of this work was to evaluate whether glycosylation might be involved in ITP ethiopathogenesis. So, in this preliminary study we aimed to evaluate the glycosylation pattern of platelets from ITP patients, its relationship with platelet features and with the response to treatments.Methods: Seventy eight ITP patients were recruited: 32 without treatment (UT-ITP) for at least six months, 35 responders to thrombopoietin receptor agonists (TPO-RA, 62.8% with eltrombopag, 27.2 % with romiplostim), and 11 refractory (r-ITP). Eighty one healthy controls were also included.Human peripheral blood samples were collected in 3.8% sodium citrate.Platelet activation was determined by flow cytometry (FCM) through binding of FITC-PAC1 (a mAb that recognizes the activated conformation of fibrinogen receptor) after activation with 100 mM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland). Apoptosis was evaluated by measurement of active caspase-3, -8 or -9 by FCM with a specific kit (Millipore, Madrid, Spain).Platelet surface exposure of lectins was analyzed with 1 µg/ml FITC-conjugated Wheat germ agglutinin lectin (WGA) or 1 µg/ml FITC-conjugated Erythrina cristagalli lectin (ECA). WGA binds to sialic acid and N-acetylglucosaminyl residues and ECA is a galactose specific legume lectin.Glycoprotein derived glycans from a healthy control and from a r-ITP patient were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion whilst O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-MS.Comparisons of quantitative variables were made with SPSS.22 software.Results: Platelet count (x109/L) was 269±55 in controls, 141±126 in UT-ITP, 111±85 in TPO-RA and 8±5 in r-ITP. ITP patients with the lowest platelet count showed the most pronounced binding of WGA and ECA (Table 1). Moreover, binding of these lectins showed a mild positive correlation with caspase activities (Table 1). No relationship was observed between platelet ability to be activated and glycosylation features.When lectins' binding was studied stratifying patients according to their response to therapeutic treatments we observed that platelets from all ITP patients bound more ECA and WGA (Figure 1). Despite no significant differences were observed between ITP groups, r-ITP seemed to bind more WGA.To increase our knowledge about protein glycosylation of platelet proteins in ITP patients we performed glycomic analyses of platelets from one healthy control and from one r-ITP patient. It was demonstrated that bi-, tri- and tetra-antennary complex type N-glycans are the dominant class of N-glycans with also some high mannose structures. Analysis of sialylated N-glycan, via digestion with sialidase, revealed lower levels of sialylation in r-ITP patient platelets compared to healthy control platelets. Platelet O-glycan data revealed the presence of mucin-type core-1 and core-2 structures.Conclusion: Glycomic analysis demonstrates that human platelet glycoproteins are modified with a complex array of both N- and O-linked glycans. Preliminary data indicates that there could be changes in glycosylation associated with immune thrombocytopenia. These changes in platelet protein glycosylation might be involved in an enhancement of apoptosis in platelets from ITP patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Bayer: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; NovoNordisk: Consultancy, Research Funding; Shire: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

A novel fucosyl glycosphingolipid of brine shrimp that is highly sensitive to endoglycoceramidase

Endoglycoceramidase (EGCase; EC 3.2.1.123) is a glycohydrolase that hydrolyzes the glycosidic linkage between the oligosaccharide and ceramide of various glycosphingolipids. We previously reported that hydra produced EGCase to digest glycosphingolipids of brine shrimp (Artemia salina), a type of aquatic crustacean used as a diet for the culture of hydra (Horibata Y, Sakaguchi K, Okino N, Iida H, Inagaki M, Fujisawa T, Hama Y, Ito M. 2004. J Biol Chem. 279:33379-33389). We report here that a major glycosphingolipid of brine shrimp is unique in structure and highly sensitive to EGCase. The glycosphingolipid was extracted from freshly hatched brine shrimp by Folch's partition, followed by mild alkaline hydrolysis and purification with a Sep-Pak plus silica cartridge. The structure of brine shrimp glycosphingolipid was determined by gas chromatography, gas chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, and (1)H-NMR spectrometry to be GlcNAcalpha1-2Fucalpha1-3Manbeta1-4Glcbeta1-1'Cer. Two major molecular species of the glycosphingolipid were identified; the sugar and sphingoid base of each were the same but the major fatty acid was C22:0 and 2-hydroxy C22:0, respectively. This is the first report describing the glycosphingolipid that has an internal fucosyl residue substituted with alpha1-2 N-acetylglucosaminyl residue. This study also suggests the biological relevance of the glycosphingolipid as a dietary source of hydra which possesses EGCase as a digestion enzyme.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm–oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and d-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar–lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm–oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose ( p < 0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

Localized Adhesion of Nongerminated Venturia inaequalis Conidia to Leaves and Artificial Surfaces

Adhesion to the host surface is the first step for successful plant pathogen development and has been reported to be associated with both passive and active processes. For conidia of Venturia inaequalis, which depend on leaf wetness for germination, this process has not yet been described. Conidia of V. inaequalis adhered to wet hydrophobic surfaces immediately after contact to the surface, hours before initiation of germination. Attachment of nongerminated conidia was much better on hydrophobic surfaces, such as apple leaves and polystyrene, than on hydrophilic glass. Conidia released adhesive material localized in a droplet named spore tip glue (STG) at the spore apex which interacted with a contact surface only when water was present. Histochemical investigations indicated the presence of proteins and carbohydrates in STG, lectin labeling the presence of beta-galactose and N-acetylglucosaminyl residues. Transmission electron microscopy revealed two phases in the STG at the tip of dry mature conidia; as STG was present on the outer side of the intact fungal cell wall its formation should be associated with the secretion of glue through pores of the conidial wall. Surface-active substances affected the adhesion of conidia to hydrophobic surfaces stressing the importance of hydrophobic interactions. The use of protein biosynthesis inhibitors did not affect adhesion of conidia indicating that the adhesive material was preformed. It is concluded that the coincidence of STG, contact to a hydrophobic surface, and free water are essential for the adhesion of V. inaequalis conidia.

Structure of the Bacteriophage φKZ Lytic Transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Glycoproteins of drusen and drusen-like lesions

Drusen are a marker of age-related macular degeneration (AMD). Lesions similar to drusen, both in histology and their clinical appearance, are also seen in choroidal tumours, chronic inflammatory and degenerative conditions of the eye, and in mesangiocapillary glomerulonephritis type II (MCGN-II). This study aims to compare the saccharide composition of these drusen-like lesions in the various ocular pathological groups and in MCGN-II. Formalin fixed and paraffin wax embedded tissue from 21 eyes was studied. The histological diagnoses included AMD, retinal detachment, phthisis bulbi following failed retinal detachment surgery, malignant melanoma, long-standing uveitis, glaucoma and MCGN II. Glycosylation was examined using a panel of twenty biotinylated lectins and an avidin-peroxidase DAB-cobalt revealing system, with and without neuraminidase pre-treatment. High mannose, bi/tri-nonbisected and bisected complex N-glycan, N-acetyl glucosaminyl, galactosyl and sialyl residues were found to be expressed by drusen, while treatment with neuraminidase exposed subterminal N-acetyl galactosamine and galactosyl residues. Similar binding patterns were found in the various pathological groups studied. As there was no significant difference in the lectin-binding pattern in drusen in different pathologies, a common pathogenesis or at least a final common pathway for the elaboration of carbohydrate components of drusen is suggested.

Neutral N-glycan patterns of the gastropods Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica

The N-glycosylation potentials of Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica were analysed by investigation of the N-glycan structures of the skin and viscera glycoproteins by a combination of HPLC and mass-spectrometry methods. It is one of the first steps to enlarge the knowledge on the glycosylation abilities of gastropods, which may help to establish new cell culture systems, to uncover new means for pest control for some species, and to identify carbohydrate-epitopes which may be relevant for immune response. All snails analysed contained mainly oligomannosidic and small paucimannosidic structures, often terminated with 3-O-methylated mannoses. The truncated structures carried modifications by beta1-2-linked xylose to the beta-mannose residue, and/or an alpha-fucosylation, mainly alpha1,6-linked to the innermost N-acetylglucosaminyl residue of the core. Many of these structures were missing the terminal N-acetylglucosamine, which has been shown to be a prerequisite for processing to complex N-glycans in the Golgi. In some species (Planorbarius corneus and Achatina fulica) traces of large structures, terminated by 3-O-methylated galactoses and carrying xylose and/or fucose residues, were also detected. In Planorbarius viscera low amounts of terminal alpha1-2-fucosylation were determined. Combining these results, gastropods seem to be capable to produce all kinds of structures ranging from those typical in mammals through to structures similar to those found in plants, insects or nematodes. The detailed knowledge of this very complex glycosylation system of the gastropods will be a valuable tool to understand the principle rules of glycosylation in all organisms.

Lytic transglycosylases: Bacterial space-making autolysins

Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product. They are ubiquitous in bacteria which produce a compliment of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. As well, the lytic transglyosylases may have a role in pathogenesis of some bacterial species. Given their important role in bacterial cell wall metabolism, the lytic transglycosylases may present an attractive new target for the development of broad-spectrum antibiotics.

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β- d-glucosyl, β- d-xylosyl, β- d-mannosyl and β- d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β- d- N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β- d- N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β- d- N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.

Chemo-enzymatic synthesis of trimeric sialyl Lewisxpentadecasaccharide

The enzymatic synthesis of trimeric sialyl Lewisxpentadecasaccharide (6), a 15-mer, from a trimannoside precursor required six different glycosyltransferase enzymes and four nucleotide donor sugars. Three N-acetylglucosaminyl residues were transferred from UDP-N-acetylglucosamine to a trimannoside by N-acetylglucosaminyltransferases I, II, and V, respectively. Galactosylation using β(1[Formula: see text]4) galactosyltransferase and UDP-galactose gave three N-acetyl lactosamine units in nonasaccharide 4. Sialylation of 4 with α(2[Formula: see text]3) sialyltransferase and CMP-N-acetylneuraminic acid was followed by fucosylation with α(1[Formula: see text]3) fucosyltransferase and GDP-fucose giving the 15-mer 6 in mg quantities. Compound 4 was also converted to a trimeric Lewisxdodecasaccharide 12-mer with α(1[Formula: see text]3) fucosyltransferase and GDP-fucose and to a trimeric α-2,6-sialyl N-acetyllactosamine dodecasaccharide 12-mer with α(2[Formula: see text]6) sialyltransferase and CMP-N-acetylneuraminic acid. Key words: glycosyltransferases, pentadecasaccharide, sialyl Lewisx.

Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa.

Lytic transglycosylases cleave the beta,1-->4 glycosidic linkages between the N-acetylmuramoyl (MurNAc) and N-acetylglucosaminyl (GlcNAc) residues of peptidoglycan with the concomitant formation of 1,6-anhydro-N-acetylmuramyl reaction products. The genes encoding two hypothetical lytic transglycosylases were identified in the genome of Pseudomonas aeruginosa PAO1 by a BLAST search using membrane-bound lytic transglycosylase B (MltB) from Escherichia coli as the query. The two genes were amplified by PCR and cloned as fusion proteins with C-terminal hexa-His sequences. Expression studies of the two genes in E. coli in the presence of [(3)H]palmitate resulted in the labeling of only one of the two enzymes. This enzyme, named MltB, was overexpressed to form insoluble inclusion bodies. Its gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which includes Cys17, the putative site of lipidation. This MltB derivative (named sMltB) was shown to not label with [(3)H]palmitate, and it was overexpressed in soluble form. The second, nonlabeled enzyme was overexpressed in soluble form and hence was named soluble lytic transglycosylase B (SltB). Both sMltB and SltB were purified to apparent homogeneity by a combination of affinity (Ni(2+)-NTA), cation-exchange (Mono S), and gel permeation (Superdex 75) chromatographies. The reaction products released by the two enzymes from purified, insoluble peptidoglycan were characterized by a novel high-performance anion-exchange chromatography (HPAEC) assay. Both enzymes produced the same three major soluble products which were identified as anhydromuropeptides based on ESI-MS analysis (cross-linked anhydrodisaccharide-tetrasaccharide, m/z obs 1824.9; anhydrodisaccharide-pentapeptide, m/z obs 922.2; and anhydrodisaccharide-tripeptide, m/z obs 851.3. The Michaelis-Menten kinetic parameters were also determined for the two enzymes using the same insoluble peptidoglycan substrate by aminosugar compositional analysis of soluble reaction products. At pH 5.8 and in the presence of 0.1% Triton, SltB was found to be more catalytically efficient, as reflected by its k(cat)/K(M) value, than sMltB.

Microscopical examination of the localisation patterns of two novel rhodamine derivatives in normal and neoplastic colonic mucosa.

Tissue characterisation by fluorescence imaging, using exogenous fluorophores, is a promising method for cancer detection. Histochemical alterations in the composition of mucins, when neoplastic transformations occur, could be exploited to derive more selective fluoroprobes indicative of early malignant transformation. The aim of this work was to develop and examine tumour selective fluoroprobes for colon cancer diagnosis, as well as to determine the morphological components where selective dye accumulation has occurred. Two novel fluoroprobes: rhodamine B-L-leucine amide and rhodamine B-phenylboronic acid were synthesised and examined together with Mayer's mucicarmine, alexa 350-wheat germ agglutinin (WGA) and tetramethyl rhodamine-concanavalin A (ConA). Fluorescence microscopy studies were performed with deparaffinised human colon sections, using an epifluorescence microscope equipped with a colour CCD camera. The intense accumulation of the novel fluoroprobes was localised in the amorphous material in the lumen of neoplastic crypts. To gain insight into the localisation patterns, mucicarmine, alexa 350-WGA and tetramethyl rhodamine-ConA were used. Alexa 350-WGA reacted primarily with mucin secreted in the malignant crypt lumen suggesting that this material is rich in sialic acid and N-acetylglucosaminyl residues. These derivatives clearly and consistently distinguished non-neoplastic from neoplastic human colon tissue sections. The intense accumulation at the altered mucins indicates that they could be used as fluoroprobes of biochemical alterations for carcinoma detection.

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.

ヘテロ分岐シクロデキストリンの合成とその性質

Hetero-branched cyclodextrins (CDs) were synthesized by transglycosylation or the reverse action of several enzymes such as βββ-galactosidases, α-galactosidases, α-mannosidases, lysozyme and R-Nacetylhexosaminidase. Their structures were analyzed by methylation, FAB-MS, and NMR spectroscopies. β-Galactosidase and a-galactosidase from microorganisms synthesized hetero-branched CDs, of which the galactose residues were linked at side chains of the branched CDs. But these enzymes could not synthesize galactosyl-CDs, directly linked to the CD ring. However, α-galactosidase from coffee bean and α-mannosidase and N-acetylhexosaminidase from jack bean could bind galactosyl, mannosyl and N-acetylglucosaminyl residues directly to the CD rings, respectively, by transglycosylation or reverse action. The effects of the side-chain residues in branched CDs, on solubility, hemolytic activity, and inclusion reactions with 6-O-α-D-glucosyl-CDs, 6-O-α-D-galactosyl-CDs, and 6-O-α-Dmannosyl-CDs, were examined.

Reaggregation and binding of cell wall proteins from Candida albicans to structural polysaccharides

Urea or hot sodium dodecyl sulphate extracted a significant amount of the same proteins from the matrix of the cell wall of the yeast form and mycelial cells of Candida albicans. Gel filtration analysis of the urea-extracted proteins revealed that they occurred in the form of large complexes which were unaffected by up to 8 M urea. Among them, proteins en route to becoming covalently associated within the wall scaffold were identified by their reaction with specific antibodies. When urea was removed by dialysis, some of these proteins specifically reassociated into large aggregates which bound strongly with ConA, whereas others remained soluble in smaller associated products. The ability of some of these proteins to bind to the insoluble wall polysaccharides was also assessed. No self-assembling proteins were able to bind to glucans and/or chitin. Specificity of the binding to polysaccharides made of β-bound glucosyl or N-acetylglucosaminyl residues was determined by the competitive effect of several disaccharides. Whereas laminaribiose and diacetylchitobiose were strong inhibitors of protein binding to both glucan and chitin, lactose, maltose and sucrose were ineffective.

Nuclear and Cytoplasmic Glycosylation

O-GlcNAcylation is a form of cytoplasmic and nuclear glycosylation that is found on many diverse proteins of the cell including RNA polymerase II and its associated transcription factors, cytoskeletal proteins, nucleoporins, viral proteins, heat shock proteins, tumor suppressors, and oncogenes. It involves the attachment of a single, unmodified N-acetylglucosaminyl residue O-glycosidically linked to the hydroxyl groups of serine and threonine moieties of proteins. It is a highly abundant and dynamic form of posttranslational modification that appears to modulate function in a manner similar to phosphorylation. All O-GlcNAc-containing proteins are phosphoproteins that are involved in the formation of multimeric complexes, suggesting that O-GlcNAc may play a role in mediating protein-protein interactions. O-GlcNAc sites resemble phosphorylation sites and in many cases the two modifications are mutually exclusive; therefore, O-GlcNAcylation may act as an antagonist of phosphorylation and help to mediate many essential functions of the cell.

Glycosidase-catalysed oligosaccharide synthesis of di-, tri- and tetra-saccharides using the N-acetylhexosaminidase from Aspergillus oryzae and the β-galactosidase from Bacillus circulans

The N-acetylhexosaminidase from Aspergillus oryzae catalyse transfer of N-acetylglucosaminyl and N-acetylgalactosaminyl residues selectively on to the 6-OH group of N-acetylgalactosamine to give the corresponding (1→6)-linked disaccharides 3 and 6 in 26 and 38% yield, respectively. The disaccharide β-D-GlcpNAc-(1→6)-β-D-GalpNAc 3 thus synthesized acts as acceptor for transfer of a β-D-galactosyl residue from the corresponding p-nitrophenyl glycoside on to the 4-OH group of the non-reducing unit to give the ovarian cyst fluid mucin fragment β-D-Galp-(1→4)-β-GlcpNAc-(1→6)-D-GalpNAc 8 in 48% yield together with the tetrasaccharide β-D-Galp-(1→4)-β-D-Gal-(1→4)-β-D-GlcpNAc-(1→6)-D-GalpNAc 9 in 7% yield. With lactose as acceptor, the trisaccharide β-D-Galp-(1→4)-β-D-Galp-(1→4)-D-Glcp 12, a growth factor for human intestinal bifidobacteria, is produced in 52% yield.p

N-acetylglucosamine-dependent nuclear import of neoglycoproteins.

Nuclear proteins bearing O-linked N-acetylglucosaminyl residues are involved in nuclear transport and in transcriptional processes. However, the role of glycosylation remains to be determined. It was proposed that glycosylation could be involved in macromolecular complex formation or in nuclear targeting. In the present study, we show that, in digitonin-permeabilized cells, BSA substituted with beta-di-N-acetylchitobioside (GlcNAc beta 4GlcNAc) is transported from the cytosol to the nucleus in a sugar dependent manner. The process is time and ATP dependent. Under the conditions used, the nuclear import of beta-di-N-acetyl-chitobios BSA, as it has also been previously shown for the nuclear import of alpha-glucosyl BSA (Duverger et al., J. Cell Sci., 108, 1325-1332, 1995) does not require the addition of a cytosolic extract, while the nuclear import of peptidic-NLS substituted BSA does require such an addition. These results suggest that GlcNAc can act as a nuclear localization signal.