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BBA - General Subjects (v.1760, #4)

Editorial Board (pp. ii).

Sweet secret of the multicellular life by Gordan Lauc (pp. 525-526).

Carbohydrates as future anti-adhesion drugs for infectious diseases by Nathan Sharon (pp. 527-537).

Adhesion of pathogenic organisms to host tissues is the prerequisite for the initiation of the majority of infectious diseases. In many systems, it is mediated by lectins present on the surface of the infectious organism that bind to complementary carbohydrates on the surface of the host tissues. Lectin-deficient mutants often lack the ability to initiate infection. The bacterial lectins are typically in the form of elongated submicroscopic multi-subunit protein appendages, known as fimbriae (or pili). The best characterized of these are the mannose-specific type 1 fimbriae, the galabiose-specific P fimbriae and the N-acetylglucosamine-specific fimbriae of Escherichia coli. Soluble carbohydrates recognized by the bacterial surface lectins block the adhesion of the bacteria to animal cells in vitro. Aromatic α-mannosides are potent inhibitors of type 1 fimbriated E. coli, being up to 1000 times more active than MeαMan, with affinities in the nanomolar range. This is due to the presence of a hydrophobic region next to the monosaccharide-binding site of the fimbriae, as recently demonstrated by X-ray studies. Polyvalent saccharides (e.g., neoglycoproteins or dendrimers) are also powerful inhibitors of bacterial adhesion in vitro. Very significantly, lectin-inhibitory saccharides have been shown to protect mice, rabbits, calves and monkeys against experimental infection by lectin-carrying bacteria. Since anti-adhesive agents do not act by killing or arresting the growth of the pathogens, it is very likely that strains resistant to such agents will emerge at a markedly lower rate than of strains that are resistant to antibiotics. Suitable sugars also inhibit the binding to cells of carbohydrate-specific toxins, among them those of Shigella dysenteriae Type 1, and of the homologous Verotoxins of E. coli, specific for galabiose. Appropriately designed polyvalent ligands are up to six orders of magnitude stronger inhibitors of toxin binding in vitro than the monovalent ones, and they protect mice against the Shigella toxin. The above data provide clear proof for the feasibility of anti-adhesion therapy of infectious diseases, although this has not yet been successful in humans. All in all, however, there is little doubt that inhibitors of microbial lectins will in the near future join the arsenal of drugs for therapy of infectious diseases.

Keywords: Escherichia coli; Fimbriae (pili); Bacterial lectin; Combining site; Three-dimensional strcuture

Tracing the history of Galα1–4Gal on glycoproteins in modern birds by Noriko Suzuki; Michael Laskowski Jr.; Yuan C. Lee (pp. 538-546).

Galα1–4Gal is typically found in mammalian glycolipids in small quantities, and recognized by some pathogens, such as uropathogenic Escherichia coli. In contrast, glycoproteins containing Galα1–4Gal were rarely found in vertebrates except in a few species of birds and amphibians until recently. However, we had previously reported that pigeon ( Columba livia) egg white and serum glycoproteins are rich in N-glycans with Galα1–4Gal at non-reducing termini. Our investigation with egg white glycoproteins from 181 avian species also revealed that the distribution of (Galα1–4Gal)-containing glycoproteins was not rare among avians, and is correlated with the phylogeny of birds. The differentiated expression was most likely emerged at earlier stage of diversification of modern birds, but some birds might have lost the facility for the expression relatively recently.

Keywords: Abbreviations; Con A; concanavalin A; GalT; galactosyltransferase; GS-I; Griffonia simplicifolia; I; mya; million years ago; NeuAc; N; -acetylneuraminic acid; NeuGc; N; -glycolylneuraminic acid; RCA-I; Ricinus communis; agglutinin I; UDP; uridine 5′-diphosphateGlycan diversity; Modern birds; Evolution; Galabiose; Molecular defense system; Pathogenic microbes

Applied glycoproteomics—approaches to study genetic-environmental collisions causing protein-losing enteropathy by Lars Bode; Hudson H. Freeze (pp. 547-559).

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a life-threatening symptom associated with seemingly unrelated conditions including Crohn's disease, congenital disorder of glycosylation, or Fontan surgery to correct univentricular hearts. Emerging commonalities between these and other disorders led us to hypothesize that PLE develops when genetic insufficiencies collide with simultaneous or sequential environmental insults. Most intriguing is the loss of heparan sulfate (HS) proteoglycans (HSPG) specifically from the basolateral surface of intestinal epithelial cells only during PLE episodes suggesting a direct link to protein leakage. Reasons for HSPG loss are unknown, but genetic insufficiencies affecting HSPG biosynthesis, trafficking, or degradation may be involved. Here, we describe cell-based assays we devised to identify key players contributing to protein leakage. Results from these assays confirm that HS loss directly causes protein leakage, but more importantly, it amplifies the effects of other factors, e.g., cytokines and increased pressure. Thus, HS loss appears to play a central role for PLE. To transfer our in vitro results back to the in vivo situation, we established methods to assess enteric protein leakage in mice and present several genetically deficient strains mimicking intestinal HS loss observed in PLE patients. Preliminary results indicate that mice with haploinsufficient genes involved in HS biosynthesis or HSPG trafficking develop intestinal protein leakage upon additional environmental stress. Our goal is to model PLE in vitro and in vivo to unravel the pathomechanisms underlying PLE, identify patients at risk, and provide them with a safe and effective therapy.

Keywords: Abbreviations; CDG; Congenital disorder of glycosylation; DPP IV; dipeptidylpeptidase IV; GAG; glycosaminoglycans; HS; heparan sulfate; HSase; heparinase II; HSPG; heparan sulfate proteoglycan; LLO; lipid-linked oligosaccharide; PLE; protein-losing enteropathy; PMM; phosphomannose mutase; PMI; phosphomannose isomerase; TER; transepithelial resistance; TNFR1; TNFα receptor 1Protein-losing enteropathy; Heparan sulfate; TNFα; Fontan surgery; Heart defect; Congenital disorder of glycosylation

The role of O-linked and N-linked oligosaccharides on the structure–function of glycoprotein hormones: Development of agonists and antagonists by Fuad Fares (pp. 560-567).

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common α and a hormone specific β subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the α and β subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (α, FSHβ and hCGβ) asparagine-linked ( N-linked) oligosaccharides. CGβ subunit is distinguished among the β subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure–function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.

Keywords: Abbreviations; TSH; Thyrotropin; FSH; Follitropin; LH; Lutropin; hCG; human Chorionic Gonadotropin; CHO; Chinese Hamster Ovary; TSI; Thyroid-Stimulating Immunoglobulin; CTP; Carboxyl-Terminal Peptide; T; ₃; Triiodothyronine; T; ₄; Tetraiodothyronine (Thyroxine)Glycoprotein hormone; N; -linked oligosaccharide; O; -linked oligosaccharide; Agonist; Antagonist

The role of informatics in glycobiology research with special emphasis on automatic interpretation of MS spectra by Claus-W. von der Lieth; Thomas Lütteke; Martin Frank (pp. 568-577).

This paper reviews the current status of bioinformatics applications and databases in glycobiology, which are based on bioinformatics approaches as well as informatics for glycobiology where an explicit encoding of glycan structures is required. The availability of the complete sequence of the human genome has accelerated the systematic identification of so far unidentified glycogenes considerably in many areas of glycobiology using well-established bioinfomatics tools. Although there has been an immense development of new glyco-related data collections as well as informatics tools and several efforts have been started to cross-link and reference the various data deposited in distributed databases, informatics for glycobiology and glycomics is still poorly developed compared to the genomics and proteomics area. The development of algorithms for the automatic interpretation of MS spectra – currently, a severe bottleneck, which hampers the rapid and reliable interpretation of MS data in high-throughput glycomics projects – is reviewed. A comprehensive list of web resources is given. Several lines of progression are discussed. There is an urgent need for the development of decentralised input facilities of experimentally determined glycan structures. Simultaneously, agreements of standards for the structural description of glycans as well as formats for the related data have to be established. The integration of glycomics with genomics/proteomics has to increase.

Keywords: Glycobiology; Glycomics; Bioinformatics; Database; Automatic MS interpretation

Bioinformatics for comprehensive finding and analysis of glycosyltransferases by Norihiro Kikuchi; Hisashi Narimatsu (pp. 578-583).

Bioinformatics is a very powerful tool in the field of glycoproteomics as well as genomics and proteomics. As a part of the Glycogene Project (GG project), we have developed a novel bioinformatics system for the comprehensive identification and in silico cloning of human glycogenes. Using our system, a total of 105 candidate human glycogenes were identified and then engineered for heterologous expression. Of these candidates, 38 recombinant proteins were successfully identified for their enzyme activity and substrate specificity. We also classified 47 out of 60 carbohydrate-active enzyme glycosyltransferase families into 4 superfamilies using the profile Hidden Markov Model method. On the basis of our classification and the relationship between glycosylation pathways and superfamilies, we propose the evolution of glycosyltransferases.

Keywords: Abbreviations; EM; expectation maximization; EST; expressed sequence tag; ORF; open reading frame; GT; glycosyltransferase; HMM; Hidden Markov modelBioinformatics; Glycosyltransferase; Glycogene; Glycoproteomics; Evolution

Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins by Gordan Lauc; Marija Heffer-Lauc (pp. 584-602).

Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins have very different biosynthetic origin, but they have one thing in common: they are both comprised of a relatively large hydrophilic moiety tethered to a membrane by a relatively small lipid tail. Both gangliosides and GPI-anchored proteins can be actively shed from the membrane of one cell and taken up by other cells by insertion of their lipid anchors into the cell membrane. The process of shedding and uptake of gangliosides and GPI-anchored proteins has been independently discovered in several disciplines during the last few decades, but these discoveries were largely ignored by people working in other areas of science. By bringing together results from these, sometimes very distant disciplines, in this review, we give an overview of current knowledge about shedding and uptake of gangliosides and GPI-anchored proteins. Tumor cells and some pathogens apparently misuse this process for their own advantage, but its real physiological functions remain to be discovered.

Keywords: Shedding and uptake; Ganglioside; Glycosylphosphatidylinositol-anchored protein; Lipid raft; Saposin; Immunohistochemistry

Design of carbohydrate multiarrays by V.I. Dyukova; N.V. Shilova; O.E. Galanina; A.Yu. Rubina; N.V. Bovin (pp. 603-609).

Recently, microarray technology has increasingly been widely applied in glycobiology. This technology has rather evident potential advantages: unlimited number of carbohydrate ligands coated onto one small sized chip, enormously low consumption of both carbohydrate ligands and carbohydrate-binding proteins to be tested, etc. Literature data demonstrate that three approaches are used for glycoarray design. The first one is based on the physical adsorption of glycomolecules on a surface (as in a common ELISA), the second one—on covalent immobilization, and the third one—on a streptavidin–biotin system. In all of the described methods, carbohydrate ligands were placed on chips as a 2D monolayer and high sensitivity was achieved due to fluorescent detection. Notably, a tendency of stepping from model chips toward real multiarrays, where the number of carbohydrate ligands can be up to two hundred, has been observed the last 2 years, this already producing a number of interesting findings when studying carbohydrate-binding proteins.In 2005 new construction, 3D glycochip was described, where 150 μm diameter polyacrylamide gel elements serve as microreactors instead of 2D dots. As a result of the 3D placement of a ligand, two orders of magnitude increase of its density is possible, this providing principal signal improvement during fluorescent detection and increasing method sensitivity. At the same time, carbohydrate consumption is low, i.e., ∼1 pmol per gel element. Copolymerization chemistry enables the immobilization of several glycomolecule classes to the gel, in particular, aminospacered oligosaccharides, polyacrylamide conjugates, and even 2-aminopyridine derivatives of oligosaccharides, which are widely used in the structural analysis of glycoprotein N-chains.

Keywords: Glycochip; Carbohydrate array; Oligosaccharide

Remnant epitopes, autoimmunity and glycosylation by Ghislain Opdenakker; Chris Dillen; Pierre Fiten; Erik Martens; Ilse Van Aelst; Philippe E. Van den Steen; Inge Nelissen; Sofie Starckx; Francis J. Descamps; Jialiang Hu; Helene Piccard; Jo Van Damme; Mark R. Wormald; Pauline M. Rudd; Raymond A. Dwek (pp. 610-615).

The role of extracellular proteolysis in innate and adaptive immunity and the interplay between cytokines, chemokines and proteinases are gradually becoming recognized as critical factors in autoimmune processes. Many of the involved proteinases, including those of the plasminogen activator and matrix metalloproteinase cascades, and also several cytokines and chemokines, are glycoproteins. The stability, interactions with inhibitors or receptors, and activities of these molecules are fine-controlled by glycosylation. We studied gelatinase B or matrix metalloproteinase-9 (MMP-9) as a glycosylated enzyme involved in autoimmunity. In the joints of rheumatoid arthritis patients, CXC chemokines, such as interleukin-8/CXCL8, recruit and activate neutrophils to secrete prestored neutrophil collagenase/MMP-8 and gelatinase B/MMP-9. Gelatinase B potentiates interleukin-8 at least tenfold and thus enhances neutrophil and lymphocyte influxes to the joints. When cartilage collagen type II is cleaved at a unique site by one of several collagenases (MMP-1, MMP-8 or MMP-13), it becomes a substrate of gelatinase B. Human gelatinase B cleaves the resulting two large collagen fragments into at least 33 peptides of which two have been shown to be immunodominant, i.e., to elicit activation and proliferation of autoimmune T cells. One of these two remnant epitopes contains a glycan which is important for its immunoreactivity. In addition to the role of gelatinase B as a regulator in adaptive immune processes, we have also demonstrated that it destroys interferon-β, a typical innate immunity effector molecule and therapeutic cytokine in multiple sclerosis. Furthermore, glycosylated interferon-β, expressed in Chinese hamster ovary cells, was more resistant to this proteolysis than recombinant interferon-β from bacteria. These data not only prove that glycosylation of proteins is mechanistically important in the pathogenesis of autoimmune diseases, but also show that targeting of glycosylated proteinases or the use of glycosylated cytokines seems also critical for the treatment of autoimmune diseases.

Keywords: Gelatinase B/MMP-9; Remnant epitope; TIMP-1; Collagenase; Neutrophil

Galectin-3: An open-ended story by Jerka Dumic; Sanja Dabelic; Mirna Flögel (pp. 616-635).

Galectins, an ancient lectin family, are characterized by specific binding of β-galactosides through evolutionary conserved sequence elements of carbohydrate-recognition domain (CRD). A structurally unique member of the family is galectin-3; in addition to the CRD it contains a proline- and glycine-rich N-terminal domain (ND) through which is able to form oligomers. Galectin-3 is widely spread among different types of cells and tissues, found intracellularly in nucleus and cytoplasm or secreted via non-classical pathway outside of cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intra- and extracellular proteins galectin-3 affects numerous biological processes and seems to be involved in different physiological and pathophysiological conditions, such as development, immune reactions, and neoplastic transformation and metastasis. The review attempts to summarize the existing information on structural, biochemical and intriguing functional properties of galectin-3.

Keywords: Galectin-3; LGALS3; Structure; Expression; Tissue distribution; Functional properties; Immune reaction; Cancer

Glyconanoparticles: Types, synthesis and applications in glycoscience, biomedicine and material science by Jesús M. de la Fuente; Soledad Penadés (pp. 636-651).

Nanoparticles are the subject of numerous papers and reports and are full of promises for electronic, optical, magnetic and biomedical applications. Although metallic nanoparticles have been functionalized with peptides, proteins and DNA during the last 20 years, carbohydrates have not been used with this purpose until 2001. Since the first synthesis of gold nanoparticles functionalized with carbohydrates (glyconanoparticles) was reported, the number of published articles has considerably increased. This article reviews progress in the development of nanoparticles functionalized with biological relevant oligosaccharides. The glyconanoparticles constitute a good bio-mimetic model of carbohydrate presentation at the cell surface, and maybe, excellent tools for Glycobiology, Biomedicine and Material Science investigations.

Keywords: Glyconanoparticle; Quantum dot; Magnetic nanoparticle; Carbohydrate interaction; Multivalence; Glycobiology

Glycomics investigation into insulin action by Simon Parry; Dirk Hadaschik; Christine Blancher; Mande K. Kumaran; Natalia Bochkina; Howard R. Morris; Sylvia Richardson; Timothy J. Aitman; Dominique Gauguier; Ken Siddle; James Scott; Anne Dell (pp. 652-668).

Defects in glycosylation are becoming increasingly associated with a range of human diseases. In some cases, the disease is caused by the glycosylation defect, whereas in others, the aberrant glycosylation may be a consequence of the disease. The implementation of highly sensitive and rapid mass spectrometric screening strategies for profiling the glycans present in model biological systems is revealing valuable insights into disease phenotypes. In addition, glycan screening is proving useful in the analysis of knock-out mice where it is possible to assess the role of glycosyltransferases and glycosidases and what function they have at the cellular and whole organism level. In this study, we analysed the effect of insulin on the glycosylation of 3T3-L1 cells and the effect of insulin resistance on glycosylation in a mouse model. Transcription profiling of 3T3-L1 cells treated with and without insulin revealed expression changes of several glycogenes. In contrast, mass spectrometric screening analysis of the glycans from these cells revealed very similar profiles suggesting that any changes in glycosylation were most likely on specific proteins rather than a global phenomenon. A fat-fed versus carbohydrate-fed mouse insulin resistant model was analysed to test the consequences of chronic insulin resistance. Muscle and liver N-glycosylation profiles from these mice are reported.

Keywords: Glycomics; Insulin resistance; 3T3-L1; Glycogene

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis by M. Holland; H. Yagi; N. Takahashi; K. Kato; C.O.S. Savage; D.M. Goodall; R. Jefferis (pp. 669-677).

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, ∼20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.

Keywords: IgG-Fc/IgG-Fab glycosylation; IgG, ANCA; Post-translational modifications

Demonstration of the expression and the enzymatic activity of N-acetylglucosaminyltransferase IX in the mouse brain by Kei-ichiro Inamori; Satoka Mita; Jianguo Gu; Yoko Mizuno-Horikawa; Eiji Miyoshi; James W. Dennis; Naoyuki Taniguchi (pp. 678-684).

We recently reported on a brain-specific β1,6- N-acetylglucosaminyltransferase IX (GnT-IX, also referred to as GnT-VB), a GnT-V homologue, which acts on α-linked mannose of N-glycans and O-mannosyl glycans. To distinguish functions of GnT-IX with GnT-V, we examined the distribution of GnT-IX and GnT-V transcripts in mouse tissues by Northern blot analysis. The two enzymes were differentially expressed as has previously been observed in human tissues. GnT-IX transcripts were restricted to the cerebrum, cerebellum, thymus and testis, whereas GnT-V transcripts were expressed ubiquitously in mouse tissues. To investigate the localization of these enzymes in mouse tissues in more detail, a polyclonal antibody against GnT-IX was prepared. The antibody specifically recognized GnT-IX, but not GnT-V, in the Golgi apparatus, as confirmed by the use of GnT-IX and GnT-V transfectants. In agreement with the Northern blot analysis data, an immunohistochemical study showed substantial expression of GnT-IX in the brain, while no expression was observed in the liver. Moreover, to exclude GnT-V contamination, we performed an enzymatic assay for GnT-IX using a Mgat5 (GnT-V)-null mouse brain as an enzyme source and found the enzymatic activities do, in fact, exist in mouse brain. The reaction product was confirmed by high performance liquid chromatography and mass spectrometry. These results demonstrate, for the first time, that GnT-IX protein is actually expressed and may function as a glycosyltransferase in the brain.

Keywords: GnT-IX; GnT-V; Mgat5; N-glycan; Brain

Probing the substrate specificity of four different sialyltransferases using synthetic β-d-Gal p-(1→4)-β-d-Glc pNAc-(1→2)-α-d-Man p-(1→O) (CH₂)₇CH₃ analogues by Philippe F. Rohfritsch; John A.F. Joosten; Marie-Ange Krzewinski-Recchi; Anne Harduin-Lepers; Benoit Laporte; Sylvie Juliant; Martine Cerutti; Philippe Delannoy; Johannes F.G. Vliegenthart; Johannis P. Kamerling (pp. 685-692).

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of β-d-Gal p-(1→4)-β-d-Glc pNAc-(1→2)-α-d-Man p-(1→O)(CH₂)₇CH₃ analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminald-GlcN were tested for their influence on the α-2,3- and α-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers β-d-Gal pNAc-(1→4)-β-d-Glc pNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.

Keywords: Abbreviations; Alt; p; L-altropyranosyl; CMP-Neu5Ac; cytidine monophosphate; N; -acetylneuraminic acid; Gal; p; D-galactopyranosyl; Glc; p; NAc; N; -acetyl-; d; -glucosaminopyranosyl; Gul; p; D-gulopyranosyl; Man; p; D-mannopyranosyl; Neu5Ac; N; -acetylneuraminic acid (sialic acid); ST3Gal; CMP-Neu5Ac: Galβ1-R α-2,3-sialyltransferase; ST6Gal; CMP-Neu5Ac: Galβ1-R α-2,6-sialyltransferaseSialyltransferases; Substrate specificity; Synthetic analogue

Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy by Yoshiki Yamaguchi; Mamiko Nishimura; Mayumi Nagano; Hirokazu Yagi; Hiroaki Sasakawa; Kazuhisa Uchida; Kenya Shitara; Koichi Kato (pp. 693-700).

The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C_H2 domain of each heavy chain with microheterogeneities depending on physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fcγ receptor (FcγR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcγRIIIa (sFcγRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFcγRIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgG1-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgG1-Fc for sFcγRIIIa. On the other hand, those glycosidase treatments did not significantly affect the affinity of IgG1-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the C_H2 domains. Furthermore, the cleavage at the GlcNAcβ1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major FcγR-binding site, while the conformation of the C_H2/C_H3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the FcγR-binding site.

Keywords: Abbreviations; ADCC; antibody-dependent cellular cytotoxicity; CHO; Chinese hamster ovary; C1q; component of the C1 complex of the classical component cascade; Endo D; endoglycosidase D; HSQC; heteronuclear single-quantum correlation; FcγR; Fcγ receptor; NMR; nuclear magnetic resonance; PNGase F; peptide:; N; -glycanase F; RU; resonance units; SPR; surface plasmon resonanceIgG; Fc; Glycoprotein; Glycoform; Fcγ receptor; NMR

Galectin-3 in macrophage-like cells exposed to immunomodulatory drugs by Sanja Dabelic; Sandra Supraha; Jerka Dumic (pp. 701-709).

During the last few decades, the effects of immunomodulatory drugs on numerous molecules and biological processes have been widely studied. Nevertheless, the relationship between immunomodulatory drugs and lectin expression/function is still to be elucidated. In this study, we used THP-1-derived macrophages to investigate the effects of non-steroidal anti-inflammatory drugs (aspirin and indomethacin) and glucocorticoids (hydrocortisone and dexamethasone) on galectin-3, a multifunctional β-galactoside binding lectin, which in general acts as a strong pro-inflammatory signal. The results showed that all immunomodulatory drugs applied in clinically relevant doses affect both the gene ( LGALS3) and protein expression level of galectin-3. The provoked changes on protein level are qualitatively and quantitatively different comparing to the effects on galectin-3 mRNA level, and depend on the differentiation state of the cell, drug type and applied concentration as well as on time of the exposure. Our data revealed galectin-3 as a new target molecule of immunomodulatory drugs, thus suggesting an additional pathway of their action on immune response.

Keywords: Abbreviations; ASA; aspirin; Dex; dexamethasone; DMSO; dimethyl-sulphoxide; gal-3; galectin-3; HC; hydrocortisone; HRP; horse radish peroxidase; Ind; indomethacin; LGALS3; lectin, galactoside-binding, soluble (galectin-3 gene); NSAID; non-steroidal anti-inflammatory drug; PMA; phorbol-12-myristate-13-acetate; RT-PCR; reverse transcription polymerase chain reactionGalectin-3; Glucocorticoids; LGALS3; Non-steroidal anti-inflammatory drugs; Macrophage

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis by Tamara L. Barron; Salvatore J. Turco (pp. 710-714).

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.

Keywords: Abbreviations; LPG; lipophosphoglycan; GPI; glycosylphosphatidylinositol; CE; capillary electrophoresis; APTS; 8-aminopyrene-1, 3,6-trisulfonic acid Leishmania; Lipophosphoglycan; Capillary electrophoresis

Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry by Daniel Kolarich; Friedrich Altmann; Elumalai Sunderasan (pp. 715-720).

The lecithinase homolog (Hev b 4) from Hevea brasiliensis (Q6T4P0_HEVBR) is an important natural rubber latex allergen. Hev b 4 is a highly glycosylated protein and its carbohydrate moiety has been implicated in the binding of IgE from natural rubber latex allergic patients. The cDNA for Hev b 4 has recently been cloned and sequenced. Here, we have analyzed the post-translational modifications of natural Hev b 4 by liquid chromatography/electrospray ionization-mass spectrometry of tryptic peptides. Seven of the eight potential glycosylation sites were found to be occupied. One site, however, was only partially glycosylated. Asn224 was substituted by complex type N-glycans with fucose and xylose, whereas all other sites carried either oligomannose glycans or a mixture of oligomannose and complex N-glycans. Glycosylation site Asn308, the most C-terminal one of the eight sites, was only found in the non-glycosylated form. The complex type N-glycans apparently form the molecular basis for the immune reaction with patients' sera. A large fraction of Hev b 4 molecules contains two or more complex N-glycans and thus a physiological reaction against these polyvalent allergens on the basis of the carbohydrate is in theory possible. Aside from allowing glycosylation analysis, the mass spectrometric data defined the N-terminal cleavage site of Hev b 4. This study once more demonstrates the outstanding analytical potential of electrospray ionization-mass spectrometry coupled with liquid chromatographic separation.

Keywords: CD; cross-reactive carbohydrate determinant; Man5; Man6 etc., oligomannosidic N-glycans with 5, 6 etc. mannose residues; MMXF; ³; Manα-6(Manα-3)Manβ-4GlcNAcβ-4(Fucα-3)GlcNAc- (see also Fig. 1); MGnXF; ³; Manα-6(GlcNAcβ-2Manα-3)Manβ-4GlcNAcβ-4(Fucα-3)GlcNAc-; GnMXF; ³; GlcNAcβ-2Manα-6(Manα-3)Manβ-4GlcNAcβ-4(Fucα-3)GlcNAc-; GnGnXF; ³; GlcNAcβ-2Manα-6(GlcNAcβ-2Manα-3)Manβ-4GlcNAcβ-4(Fucα-3)GlcNAc- (see also http://www.proglycan.com for an explanation of N-glycan abbreviations); LC/ESI-MS; liquid chromatography/electrospray ionization-mass spectrometry; MALDI-TOF MS; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NRL; natural rubber latex; Q-TOF; quadrupol time-of-flightNatural rubber latex; Latex allergy; Hevea brasiliensis; Glycoprotein; Glycoproteomics; ESI-MS

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BBA - General Subjects (v.1760, #4)