Showing papers in "Glycobiology in 2002"
TL;DR: The aim of this review is to describe the glycopeptide linkages that have been found to date and specify their presence on well-characterized glycoproteins and with a number of human disease states in which defects in enzymatic transfer of saccharides to protein have been implicated.
Abstract: Formation of the sugar-amino acid linkage is a crucial event in the biosynthesis of the carbohydrate units of glycoproteins. It sets into motion a complex series of posttranslational enzymatic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biological functions. These reactions occur throughout the entire phylogenetic spectrum, ranging from archaea and eubacteria to eukaryotes. It is the aim of this review to describe the glycopeptide linkages that have been found to date and specify their presence on well-characterized glycoproteins. A survey is also made of the enzymes involved in the formation of the various glycopeptide bonds as well as the site of their intracellular action and their affinity for particular peptide domains is evaluated. This examination indicates that 13 different monosaccharides and 8 amino acids are involved in glycoprotein linkages leading to a total of at least 41 bonds, if the anomeric configurations, the phosphoglycosyl linkages, as well as the GPI (glycophosphatidylinositol) phosphoethanolamine bridge are also considered. These bonds represent the products of N- and O-glycosylation, C-mannosylation, phosphoglycation, and glypiation. Currently at least 16 enzymes involved in their formation have been identified and in many cases cloned. Their intracellular site of action varies and includes the endoplasmic reticulum, Golgi apparatus, cytosol, and nucleus. With the exception of the Asn-linked carbohydrate and the GPI anchor, which are transferred to the polypeptide en bloc, the sugar-amino acid linkages are formed by the enzymatic transfer of an activated monosaccharide directly to the protein. This review also deals briefly with glycosidases, which are involved in physiologically important cleavages of glycopeptide bonds in higher organisms, and with a number of human disease states in which defects in enzymatic transfer of saccharides to protein have been implicated.
1,309 citations
TL;DR: Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.
Abstract: Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to TGFbetas and collagens and other matrix molecules. In vitro, SLRPs were shown to regulate collagen fibrillogenesis, a process essential in development, tissue repair, and metastasis. To better understand their functions in vivo, mice deficient in one or two of the four most prominent and widely expressed SLRPs (biglycan, decorin, fibromodulin, and lumican) were recently generated. All four SLRP deficiencies result in the formation of abnormal collagen fibrils. Taken together, the collagen phenotypes demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix. In addition, SLRP-deficient mice develop a wide array of diseases (osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal diseases), most of them resulting primarily from an abnormal collagen fibrillogenesis. The development of these diseases by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors for these diseases. Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.
430 citations
TL;DR: Dermatan and dermatan sulfate proteoglycans have also been implicated in cardiovascular disease, tumorigenesis, infection, wound repair, and fibrosis as mentioned in this paper, and growing evidence suggests that this glycosaminoglycan is an important cofactor in a variety of cell behaviors.
Abstract: Glycosaminoglycans constitute a considerable fraction of the glycoconjugates found on cellular membranes and in the extracellular matrix of virtually all mammalian tissues. Their ability to bind and alter protein-protein interactions or enzymatic activity has identified them as important determinants of cellular responsiveness in development, homeostasis, and disease. Although heparan sulfate tends to be emphasized as the most biologically active glycosaminoglycan, dermatan sulfate is a particularly attractive subject for further study because it is expressed in many mammalian tissues and it is the predominant glycan present in skin. Dermatan and dermatan sulfate proteoglycans have also been implicated in cardiovascular disease, tumorigenesis, infection, wound repair, and fibrosis. Growing evidence suggests that this glycosaminoglycan, like the better studied heparin and heparan sulfate, is an important cofactor in a variety of cell behaviors.
418 citations
TL;DR: In this paper, the onset of nodule development, the result of rhizobia-legume symbioses, is determined by the exchange of chemical compounds between microsymbiont and leguminous host plant.
Abstract: The onset of nodule development, the result of rhizobia-legume symbioses, is determined by the exchange of chemical compounds between microsymbiont and leguminous host plant. Lipo-chitooligosaccharidic nodulation (Nod) factors, secreted by rhizobia, belong to these signal molecules. Nod factors consist of an acylated chitin oligomeric backbone with various substitutions at the (non)reducing-terminal and/or nonterminal residues. They induce the formation and deformation of root hairs, intra- and extracellular alkalinization, membrane potential depolarization, changes in ion fluxes, early nodulin gene expression, and formation of nodule primordia. Nod factors play a key role during nodule initiation and act at nano- to picomolar concentrations. A correct chemical structure is required for induction of a particular plant response, suggesting that Nod factor-receptor interaction(s) precede(s) a Nod factor-induced signal transduction cascade. Current data on Nod factor structures and Nod factor-induced responses are highlighted as well as recent advances in the characterization of proteins, possibly involved in recognition of Nod factors by the host plant.
378 citations
TL;DR: Understanding how galectins regulate cell viability and function will broaden the knowledge of the roles of galectin in basic biological processes and facilitate development of therapeutic applications for galECTins in autoimmunity, transplant-related disease, and cancer.
Abstract: Control of cell death is critical in eukaryotic development, immune system homeostasis, and control of tumorigenesis. The galectin family of lectins is implicated in all of these processes. Other families of molecules function as death receptors or death effectors, but galectins are uniquely capable of acting both extracellularly and intracellularly to control cell death. Extracellularly, galectins cross-link glycan ligands to transduce signals that lead directly to death or that influence other signals regulating cell fate. Intracellular expression of galectins can modulate other signals controlling cell viability. Individual galectins can act on multiple cell types, and multiple galectins can act on the same cell. Understanding how galectins regulate cell viability and function will broaden our knowledge of the roles of galectins in basic biological processes and facilitate development of therapeutic applications for galectins in autoimmunity, transplant-related disease, and cancer.
216 citations
TL;DR: The data indicate that the fructo-oligosaccharides accumulated in many plant species under stress conditions could indeed play an important role in cellular dehydration tolerance.
Abstract: The fructan family of oligo- and polysaccharides is a group of molecules that have long been implicated as protective agents in the drought and freezing tolerance of many plant species. However, it has been unclear whether fructans have properties that make them better protectants for cellular structures than other sugars. We compared the effects of fructans and glucans on membrane stability during air-drying. Although glucans of increasing chain length were progressively less able to stabilize liposomes against leakage of aqueous content after rehydration, fructans showed increased protection. On the other hand, glucans became more effective in protecting liposomes against membrane fusion with increasing chain length, whereas fructans became less effective. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature (T(m)) of air-dried liposomes by approximately 25 degrees C in the presence of sucrose and maltose. For the respective pentasaccharides, the reduction of T(m) of the lipids was 9 degrees C lower for samples containing fructan than for those containing glucan, indicating increased sugar--membrane interactions for the fructan compared to the glucan. A reduced interaction of the longer-chain glucans and an increased interaction of the respective fructans with the phospholipid head groups in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum. Collectively, our data indicate that the fructo-oligosaccharides accumulated in many plant species under stress conditions could indeed play an important role in cellular dehydration tolerance.
205 citations
TL;DR: Experimental studies in animal models have documented a crucial role for hyaluronan in tumor growth and metastasis, and cellular manipulations have shown that hyaluranan promotes anchorage-independent growth and invasiveness, hallmarks of the malignant phenotype.
Abstract: Hyaluronan is a high-molecular-weight, negatively charged polysaccharide with unusual physical and interactive properties. Hyaluronan is localized in the extracellular matrix, at the cell surface, and inside cells. Its tissue distribution is ubiquitous, but it is particularly concentrated in pericellular matrices surrounding proliferating and migrating cells. Hyaluronan contributes to cell behavior in at least three ways. Its unique physical properties influence the biomechanical properties of extracellular and pericellular matrices; it is a template for assembly of other pericellular macromolecules; and it interacts directly with cell surface receptors that transduce intracellular signals. Experimental studies in animal models have documented a crucial role for hyaluronan in tumor growth and metastasis. Cellular manipulations have shown that hyaluronan promotes anchorage-independent growth and invasiveness, hallmarks of the malignant phenotype.
204 citations
TL;DR: It is shown that intracellular lactose was efficiently glycosylated when genes of Glycosyltransferase that use lactose as acceptor were expressed and it was also shown that exogenous NeuAc and lactose produced sialyllactose from exogenousNeuAcand lactose.
Abstract: When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc) When the beta 1,4 galactosyltransferase lgtB gene of N meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1) In a similar way, the nanA(-) E coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose
195 citations
TL;DR: The results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.
Abstract: We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.
173 citations
TL;DR: Current views on the enzymology, structures, potential evolution, and the roles of the known glycosyltransferases from Streptococcus, Pasteurella, and Escherichia are discussed.
Abstract: Glycosaminoglycans, a class of linear polysaccharides composed of repeating disaccharide units containing a hexosamine, are important carbohydrates found in many organisms. Vertebrates utilize glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Certain pathogenic bacteria produce extracellular capsules composed of glycosaminoglycans or glycosaminoglycan-like polymers that enhance the microbes' ability to infect or to colonize the host. In the period from 1993 to 2001, bacterial enzymes were discovered that catalyze the polymerization of the repeating unit of hyaluronan, chondroitin, or N-acetylheparosan (unsulfated, unepimerized heparin). Depending on the specific carbohydrate and the microorganism, either a dual-action enzyme (synthase) that transfers two distinct monosaccharides or a pair of single-action transferases are utilized to synthesize the glycosaminoglycan polymer. Current views on the enzymology, structures, potential evolution, and the roles of the known glycosyltransferases from Streptococcus, Pasteurella, and Escherichia are discussed.
138 citations
TL;DR: These two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body.
Abstract: The high-molecular-mass salivary mucin MG1, one of two major mucins produced by human salivary glands, plays an important role in oral health by coating the tooth surface and by acting as a bacterial receptor. Here this mucin was purified from the submandibular/sublingual saliva of a blood group O individual. The presence of MUC5B as the major mucin in this preparation was confirmed by amino acid analysis and its reactivity with the monoclonal antibody PAN H2. To structurally characterize MG1 carbohydrates the O-glycans were released by reductive beta-elimination. Nuclear magnetic resonance spectroscopy of the nonfractionated mixture showed that (1) fucose was present in blood group H, Le(a), Le(x), Le(b), and Le(y) epitopes; (2) NeuAc was mainly linked alpha2-3 to Gal or alpha2-6 to GalNAcol; and (3) the major internal structures were core 1 and core 2 sequences. After this preliminary analysis the released oligosaccharides were separated into neutral (56%), sialylated (26%), and sulfated (19%) fractions, with an average length of 13, 17, and 41 sugar residues, respectively. Gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of mixtures of neutral and sialylated oligosaccharides revealed at least 62 neutral and 25 sialylated oligosaccharides consisting of up to 20 monosaccharide residues. These results showed that the MG1-derived oligosaccharides were much longer than those of MG2, and only a few species were found on both molecules. Thus, these two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body. (Less)
TL;DR: The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectIn-9 from JurKat cells.
Abstract: Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA) Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9 When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9 The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells
TL;DR: Results from both heterodikaryon assays suggest that galectin-3 can exit one nucleus, travel through the cytoplasm, and enter the second nucleus, matching the definition of shuttling.
Abstract: In previous studies, we documented that galectin-3 (M(r) approximately 30,000) is a pre-mRNA splicing factor. Recently, galectin-3 was identified as a component of a nuclear and cytoplasmic complex, the survival of motor neuron complex, through its interaction with Gemin4. To test the possibility that galectin-3 may shuttle between the nucleus and the cytoplasm, human fibroblasts (LG-1) were fused with mouse fibroblasts (3T3). The monoclonal antibody NCL-GAL3, which recognizes human galectin-3 but not the mouse homolog, was used to monitor the localization of human galectin-3 in heterodikaryons. Human galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Addition of the antibiotic leptomycin B, which inhibits nuclear export of galectin-3, decreased the percentage of heterodikaryons showing human galectin-3 in both nuclei. In a parallel experiment, mouse 3T3 fibroblasts, which express galectin-3, were fused with fibroblasts derived from a mouse in which the galectin-3 gene was inactivated. Mouse galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Again, addition of leptomycin B restricted the presence of galectin-3 to one nucleus of a heterodikaryon. The results from both heterodikaryon assays suggest that galectin-3 can exit one nucleus, travel through the cytoplasm, and enter the second nucleus, matching the definition of shuttling.
TL;DR: This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.
Abstract: The proteoglycans, especially the large chondroitin sulfate proteoglycan aggrecan, have long been viewed as important components of the extracellular matrix of cartilage. The drastic change in expression during differentiation from mesenchyme to cartilage, the loss of tissue integrity associated with proteoglycan degradation in several disease processes and, most important, the demonstration of abnormalities in proteoglycan production concomitant with the aberrant growth patterns exhibited by the brachymorphic mouse, the cartilage matrix deficient mouse, and the nanomelic chick provide the strongest evidence that the proteoglycan aggrecan is essential during differentiation and for maintenance of the skeletal elements. More recently, mutations associated with proteoglycans other than aggrecan, especially the heparan sulfate proteoglycans, glypican and perlecan, suggest an important role for these molecules in skeletal development as well. This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.
TL;DR: The data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.
Abstract: Over the past few years it has emerged that O-mannosyl glycans are not restricted to yeasts and fungi but are also present in higher eukaryotes, including humans. They play a substantial role in the onset of muscular dystrophy and neuronal migration disorders, like muscle-eye-brain disease. Protein O-mannosyltransferase genes (PMTs) are evolutionarily conserved from yeast to human; however, little is known about these enzymes in higher eukaryotes. In this study, we cloned the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2). A detailed characterization of the mammalian POMT2, with emphasis on mouse Pomt2, shows that mammalian POMT2 is predominantly expressed in testis tissue. Due to differential transcription initiation of the mPomt2 gene, two distinct mRNA species that vary in length are formed. The shorter transcript is present in all somatic tissues examined. Expression of the corresponding hPOMT2 cDNA in mammalian cells identified POMT2 as an integral membrane protein of the endoplasmic reticulum with an apparent molecular weight of 83 kDa. The longer mPomt2 transcript is restricted to testis and encodes a testis-specific mPOMT2 protein isoform. Using in situ hybridization and immunolocalization, we demonstrate that in testis tissue mPOMT2 localizes to maturing spermatids and is abundant within the acrosome, a sperm-specific organelle essential for fertilization. Our data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.
TL;DR: The findings suggest that the level of UDP-GlcNAc can be a critical factor in the production of beta1,6-branched oligosaccharides, for example, by tumor cells, which have been thought to be closely associated with tumor progression and metastasis.
Abstract: Human beta1,6-N-acetylglucosaminyltransferase V (GnT-V) was expressed by baculovirus-insect cell system, and the purified recombinant enzyme was kinetically characterized. The data obtained were used to establish the kinetic basis of the substrate specificity toward donor nucleotide sugars, and also revealed that K(m) values for the donors are much higher compared to those of other GlcNAc transferases, the kinetic properties of which have been reported. Because this exceptionally higher K(m) suggests that GnT-V is physiologically present at far from saturated conditions, it would appear that the production of beta1,6-branched oligosaccharide, which is formed by GnT-V, could be regulated in vivo by the concentration of the donor, UDP-GlcNAc, as well as the expression levels of the enzyme. When B16 melanoma cells, which express high levels of GnT-V, were incubated with GlcNAc, the beta1,6-branched oligosaccharide levels were increased, as judged by a lectin blot analysis, in conjunction with an increase in intracellular UDP-GlcNAc. These findings suggest that the level of UDP-GlcNAc can be a critical factor in the production of beta1,6-branched oligosaccharides, for example, by tumor cells, which have been thought to be closely associated with tumor progression and metastasis.
TL;DR: The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.
Abstract: Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy. Isolated cyst wall material contains carbohydrate and protein in a ratio of 3:2 (w/w), and the carbohydrate moiety is composed of a beta(1-3)-N-acetyl-D-galactopyranosamine homopolymer. Conformational analysis by molecular dynamics and persistence length calculations of GalNAc oligomers in solution demonstrated a flexible structure consisting of left- and right-handed helical elements. It is most likely that in the solid state, the polysaccharide forms ordered helices or possibly multiple helical structures having strong interchain interactions. The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.
TL;DR: These pure, size-uniform, and large range of hyaluronan oligosaccharides will be available for investigating important biological functions of HA, such as for the determination of the size(s) of HA oligosACcharides that induce angiogenesis or mediate inflammatory responses, and to interact with HA-binding proteins and receptors both in in vitro and in vivo studies.
Abstract: Hyaluronan (HA) was depolymerized by partial digestion with testicular hyaluronidase and separated into size-uniform HA oligosaccharides from 4-mers to 52-mers by anion exchange chromatography after removal of the hyaluronidase. The purity and size of each HA oligosaccharide was confirmed by using HPLC analyses, FACE, and ESI-MS. (1)H and (13)C NMR assignments and elemental analyses were obtained for each HA oligosaccharide. Endotoxins, proteins, and DNA were absent or in trace amounts in these HA oligosaccharides. Gram/mg-scale hyaluronan oligosaccharides were obtained from 200 g of HA starting material. These pure, size-uniform, and large range of HA oligosaccharides will be available for investigating important biological functions of HA, such as for the determination of the size(s) of HA oligosaccharides that induce angiogenesis or mediate inflammatory responses, and to interact with HA-binding proteins and receptors both in in vitro and in vivo studies.
TL;DR: Results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.
Abstract: Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.
TL;DR: The results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.
Abstract: Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.
TL;DR: It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.
Abstract: Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex 7 - 9 GlcNAc 2 , whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex 8 GlcNAc 2 with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.
TL;DR: Results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.
Abstract: Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.
TL;DR: It is demonstrated that the glucose is added in O-linkage to the EGF repeat, and it is identified and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O- glucosyltransferase.
Abstract: O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.
TL;DR: A model is proposed in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.
Abstract: Heparan sulfates (HSs) exert critical regulatory actions on many proteins, including growth factors, and are essential for normal development. Variations in their specific sulfation patterns are known to regulate binding and signaling of fibroblast growth factors (FGFs) via tyrosine kinase receptors (FGFRs). We previously reported differences in sulfation patterns between HS species expressed by embryonic day 10 (E10) and E12 mouse neural precursor cells. We have examined the abilities of the different HS species to support signaling of the relevant FGF-FGFR combinations expressed early during brain development. For FGF8, which only functions early (E8-E11), E10 HS showed preferential activation. The most potent signaling for FGF8 was via FGFR3c, for which E10 HS was strongly active and E12 HS had no activity. For FGF2, which functions from E10 to E13, HS from both stages showed similar activity and were more potent at activating FGFR1c than the other receptors. Thus, we find a stage-specific correlation with activation. To explore the potential mechanisms for the generation of these stage-specific HS species, we investigated the expression of the HS sulfotransferase (HSST) isozymes responsible for creating diverse sulfation motifs in HS chains. We find that there are stage-specific combinations of HSST isozymes that could underlie the synthesis of different HS species at E10 and E12. Collectively, these data lead us to propose a model in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.
TL;DR: This report is to the authors' knowledge the first demonstration of a fucosidase that can efficiently release alpha-L-fucose from fucoidan.
Abstract: Algal fucoidan is an alpha-L-fucose-based polysaccharide endowed with important biological properties for which the structure has not yet been fully elucidated. In an attempt to implement new enzymatic tools for structural study of this polysaccharide, we have found a fucosidase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on a fucoidan extracted from the brown algae Ascophyllum nodosum. We now report the purification and characterization of this alpha-L-fucosidase (EC 3.2.1.51). The enzyme was purified by three chromatographic steps, including an essential affinity chromatography based on the glycosidase inhibitor analog 6-amino-deoxymannojirimycin as the ligand. The purified alpha-L-fucosidase is a tetrameric glycoprotein of 200 kDa that hydrolyzes the synthetic substrate p-nitrophenyl alpha-L-fucopyranoside with a K(m) value of 650 microM. This enzyme has high catalytic activity (85 micromol x min(-1) x mg(-1)) compared with the other known fucosidases and also possesses an unusual thermal stability. The purified alpha-L-fucosidase is a retaining glycosidase. The activity of the purified fucosidase was determined on two structurally different fucoidans of the brown algae A. nodosum and Fucus vesiculosus to delineate glycosidic bond specificity. This report is to our knowledge the first demonstration of a fucosidase that can efficiently release alpha-L-fucose from fucoidan.
TL;DR: The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis.
Abstract: The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis. These key precursors are vital in the biosynthesis of many essential aspects of the mycobacterial cell wall. Rv2682c encodes the conserved DRAG sequence that has been proposed as a signature motif for DXSs and also all 13 conserved amino acid residues thought to be important to the function of transketolase enzymes. Recombinant Rv2682c is capable of utilizing glyceraldehyde 3-phosphate and erythrose 4-phosphate as well as D- and L-glyceraldehyde as aldose substrates. The enzyme has K(m) values of 40 microM, 6.1 microM, 5.6 mM, and 4.5 mM for pyruvate, D-glyceraldehyde 3-phosphate, D-glyceraldehyde, and L-glyceradehyde, respectively. Rv2682c has an absolute requirement for divalent cation and thiamin diphosphate as cofactors. The K(d) (thiamin diphosphate )for the native M. tuberculosis DXS activity partially purified from M. tuberculosis cytosol is 1 microM in the presence of Mg(2+).
TL;DR: A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease and was efficiently incorporated into fibroblasts derived from patients withFabry disease.
Abstract: A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.
TL;DR: There is increasing evidence that the function of agrin, a synaptogenic factor in neuromuscular formation, is modulated by several glycans, and the recent generation of ST8SiaIV null mice strongly suggests a role for polysialic acid in synaptic plasticity in the some regions of the central nervous system.
Abstract: Synapses are the fundamental units of connectivity that link together the nervous system. Lectin studies from 30 years ago suggested that specific glycans are concentrated at neuromuscular synapses in the peripheral nervous system and at excitatory synapses in the brain. Subsequent studies have confirmed that particular glycan structures are localized at these synapses, including polysialic acid, high mannose, the cytotoxic T cell antigen, and forms of heparan sulfate. Though the role of these molecules in synapse formation and function is still poorly understood, there is increasing evidence that the function of agrin, a synaptogenic factor in neuromuscular formation, is modulated by several glycans. In addition, the recent generation of ST8SiaIV null mice strongly suggests a role for polysialic acid in synaptic plasticity in the some regions of the central nervous system.
TL;DR: This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.
Abstract: We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.
TL;DR: A structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions are observed.
Abstract: The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual alpha 2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N-treated PSA-NCAM was separated by SDS-PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)(2)(Man)(5) up to (GlcNAc)(2)(Man)(9). In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.