Showing papers in "Glycobiology in 1996"

Brain extracellular matrix.

Abstract: The extracellular matrix of the adult brain tissue has a unique composition. The striking feature of this matrix is the prominence of lecticans, proteoglycans that contain a lectin domain and a hyaluronic acid-binding domain. Hyaluronic acid and tenascin family adhesive/anti-adhesive proteins are also abundant. Matrix proteins common in other tissues are nearly absent in adult brain. The brain extracellular matrix appears to have trophic effects on neuronal cells and affect neurite outgrowth. The unique composition of this matrix may be responsible for the resistance of brain tissue toward invasion by tumors of non-neuronal origin.

Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies.

Abstract: Arabinogalactan-proteins (AGPs) are a diverse class of plant cell surface proteoglycans implicated in a range of fundamental processes associated with plant cell development. Anti-AGP monoclonal antibodies have been used extensively for the investigation of the developmental regulation of AGPs although virtually nothing is known about the structure of the carbohydrate epitopes recognised by these antibodies. In this report, a series of methyl glycosides of monosaccharides and a range of oligosaccharides that are elements of the carbohydrate component of AGPs have been investigated for recognition by previously derived anti-AGP monoclonal antibodies. No clear evidence was obtained for the involvement of terminal arabinofuranosides, nor of the galactan backbone, in the recognition of the glycan structure of AGPs by any of the antibodies used in this study. Interestingly, the most effective inhibitor of the binding of the monoclonal antibodies MAC207, JIM4 and JIM13 to exudate gum antigens was an acidic trisaccharide, isolated from a partial acid hydrolysate of gum karaya which has the structure: GlcA beta(1-->3) GalA alpha(1-->2)Rha, determined by a combination of FAB-MS, GC-MS and NMR spectroscopy.

A family of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferases control the initiation of mucin-type O-linked glycosylation

Abstract: Enzymatic glycosylation of proteins involves the addition of a monosaccharide or, in the case of N-linked glycosylation of asparagine, a preformed oligosaccharide to an amino acid in a given protein. The initial step of protein glycosylation is an important event in the formation of a given glycopeptide linkage (glycoconjugate type), which involves essential recognition events between the protein and glycosyltransferase that determine the specific sites of glycan attachment. Complete processing and extension of glycan chains involves the cooperative action of perhaps hundreds of different glycosyltransferases, which successively add monosaccharides to the growing glycan chain. The characterization of glycan structures on glycoproteins as well as the identification of specific sites of glycan attachment are important for understanding the structure of a given glycoprotein, its function, and its immunobiology (Lis et al., 1993; Varki, 1993; Parekh, 1994). Mucin-type O-glycosylation is initiated by the enzyme UDP-N-acetylgalactosamine: polypeptide yV-acetylgalactosaminyltransferase (GalNAc-transferase) (EC. 2.4.1.41). In this reaction GalNAc (derived from the donor substrate UDPGalNAc) is transferred to the side chains of serine and threonine residues on polypeptides. This reaction, which has been named mucin-type O-glycosylation because of its preponderance on mucin-like glycoproteins, is one of the most abundant forms and means of glycosylation found in animals and is not restricted to mucin-like glycoproteins. This review will cover recent developments in our understanding of a family of GalNAc-transferases that mediate this type of protein glycosylation. Mucin-type glycosylation is an important post-translational modification of many animal proteins. Alterations in the Oglycosylation patterns of some proteins may play a role in the pathogenesis of several diseases in which there is evidence for altered glycoprotein structure, including ulcerative colitis, chronic bronchitis, cystic fibrosis, and cancer (Varki, 1993). In addition to its structural and functional importance to mucins, in which O-linked carbohydrates may constitute up to 80% of the total mass of these glycoproteins, O-glycosylation has been shown to be important to the folding of proteins such as hCG3, and the conformation and protease resistance of 'stem regions' of membrane-bound proteins. Mucin-like domains are also found on cell surface associated molecules functioning as selectin ligands (e.g., PSGL-1, MadCAM, CD-34; see Varki, 1993). A long-standing puzzle has been what determines sites of O-glycosylation. This is an important question because of evidence that spacing of O-glycans on many glycoproteins may be important to their function (Springer, 1994). The molecular processes governing the specificity and kinetics of mucin-type O-linked glycosylation and the parallel Man-type glycosylation of serine/threonine in yeast are poorly understood. It has not been possible thus far to determine precise positions of glycan attachment to the protein backbone for many glycoproteins. This is due, in part, to the difficulties of addressing these questions experimentally because mucins and mucin-like glycoproteins are often insensitive to protease digestion and glycans are often clustered in arrays, making sequence analysis difficult (Piller and Piller, 1993; Gooley et al, 1994). Consensus motifs for glycosylation have been described for prediction of several types of glycosylation sites other than mucin-type O-glycosylation. The best characterised example is that of N-linked glycosylation of asparagine where the consensus sequence for the acceptor site has been resolved to a short peptide sequence Asn-Xaa-Ser/Thr (where Xaa cannot be Pro) (Marshall, 1972), although Asn-Xaa-Cys has also been found to be utilized. Recent studies indicate that Asn-Xaa-Ser is less well utilized compared to the Thr containing sequon (Kasturi et al, 1995). Studies of other types of glycopeptide linkages also suggest the existence of rather specific primary peptide sequences associated with glycosylation. Thus, proteoglycantype glycosylation of serine is restricted to -Ser-Gly-Xaa-Gly(Bourdon et al, 1987), and collagen-type glycosylation of hydroxylysine restricted to -Gly-Xaa-Hyl-Gly(Prockop et al., 1979). The GlcNAc-type glycosylation of serine or threonine appears to be adjacent to an acidic amino acid and within two residues of a proline (Haltiwanger et al., 1992). The Fuc-type glycosylation of serine/threonine seems to be restricted to the peptide sequence -Gly-Gly-Thr/Ser-Cys(Harris and Spellman, 1993), and recently a prokaryotic O-linked glycan type was found associated with -Asp-Ser(Plummer et al., 1995).

Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer.

Abstract: Yeast cell wall proteins, including Cwp1p and alpha-agglutinin, could be released by treating the cell wall with either beta-1,3-or beta-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown immunologically that both beta-1,3- and beta-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and alpha-agglutinin. It was further shown that beta-1,3-glucan was linked to the wall protein through a beta-1,6-glucan moiety. The beta-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a beta-1,3-/beta-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble beta-1,3-/beta-1,6-glucan heteropolymer characterized by Fleet and Manners (1976, 1977).

Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins.

Abstract: Many plant proteins, particularly those found in foods and pollen, are known to act as sensitizing agents in humans upon repeated exposure. Among the cereal flour proteins involved in asthmatic reactions, those members of the alpha-amylase inhibitor family which are glycosylated, polypeptides, BMAI-1, BTAI-CMb*, and WTAI-CM16* are particularly reactive both in vivo and in vitro. We show here that these major glycoprotein allergens carry a single asparagine-linked complex glycan that contains both beta 1-->2 xylose and alpha 1-->3 fucose. Evidence is presented that the xylosyl residue and, to a lesser extent, the fucosyl residue are key IgE-binding epitopes and largely responsible for the allergenicity of these and unrelated proteins from plants and insects. Our results suggest that the involvement of xylose- and fucose-containing complex glycans in allergenic responses may have been underestimated previously; these glycans provide a structural basis to help explain the cross-reactivities often observed between pollen, vegetable food, and insect allergens.

Systematic nomenclature for sialyltransferases

Abstract: Laboratory for Molecular Glycobiology, Frontier Research Program, TheInstitute of Physical and Chemical Research (RIKEN), Wako, Saitama,351 01, Japan and 'Cytel Corporation and the Department of Chemistry andMolecular Biology, Scnpps Research Institute, 3525 John Hopkins Court,San Diego, CA 92121, US A

Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae

Abstract: The Saccharomyces cerevisiae alg3-1 mutant is described as defective in the biosynthesis of dolichol-linked oligosaccharides (Huffaker and Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466-7470, 1983). Man5GlcNAc2-PP-Dol accumulates in alg3 cells and Endo H resistant carbohydrates are transferred to protein by the oligosaccharyltransferase complex. In this study, we describe the cloning of the ALG3 locus by complementation of the temperature sensitive growth defect of the alg3 stt3 double mutant. The isolated ALG3 gene complements both the defect in the biosynthesis of lipid-linked oligosaccharides of the alg3-mutant and the under-glycosylation of secretory proteins. The inactivation of the nonessential ALG3 gene results in the accumulation of lipid-linked Man5GlcNac2 and protein-bound carbohydrates which are completely Endo H resistant. The ALG3 locus encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequence.

O-glcnacylation of key nuclear and cytoskeletal proteins : reciprocity with o-phosphorylation and putative roles in protein multimerization

Abstract: O-Linked N-acetylglucosamine, O-GlcNAc, was discovered (Torres and Hart, 1984) as part of our studies using highlypurified glycosyltransferases to investigate saccharide structures on lymphocytes and tumor cells (Powell et al, 1987; Whiteheart and Hart, 1987; Passaniti and Hart, 1988; Reichner et al., 1988; Whiteheart et al, 1989). Our glycosyltransferase studies are a direct outgrowth of the pioneering work of Professor Hill and his colleagues, which developed the methods for the purification of glycosyltransferases (Paulson et al., 1977; Beyer et al., 1980, 1981; Sadler et al, 1979, 1981, 1982). In particular, it was the purification (Trayer and Hill, 1971; Barkers al, 1972) and ready availability of bovine milk galactosyltransferase that allowed us to employ the enzyme as a highly specific and sensitive probe for O-GlcNAc on nuclear and cytoplasmic proteins (for methods, see Roquemore et al, 1994b). Using this enzymatic probe and other methods, we and others (for review, Hart et al, 1989, 1995a-c) have now documented that O-GlcNAc is ubiquitous and abundant on nuclear and cytoskeletal proteins of virtually all eukaryotes, including protozoans (Ortega-Barria et al, 1990; Dieckmann-Schuppert etal, 1993, 1994; Stanley etal, 1995) and fungi (Machida and Jigami, 1994). In addition, O-GlcNAc is highly dynamic, with turnover rates much higher than the protein backbones to which it is attached (Kearse and Hart, 1991; Chou et al, 1992; Roquemore et al, 19%). Virtually all O-GlcNAc proteins examined to date are also phosphoproteins, and in some instances Ser(Thr)-O-GlcNAc and Ser(Thr)-O-phosphate appear to reciprocally occupy the same hydroxyl groups (Kelly et al, 1993; Chou et al, 1995a,b). The dynamic characteristics of O-GlcNAc, and the importance of O-GlcNAcylated proteins in cellular regulation and in cytoskeletal structure, have led us to hypothesize that O-GlcNAc is a regulatory modification that not only regulates protein phosphorylation, but also is involved in modulating protein multimerization (Hart et al, 1995b,c). O-GlcNAcylation is most abundant in the nucleus

Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties

Abstract: Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.

Molecular mimicry in the recognition of glycosphingolipids by Galα3Galβ4GlcNAcβ-binding Clostridium difficile toxin A, human natural anti α-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor

Abstract: Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13. However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase. To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay. The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed. A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes. This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source. Two additional binding-active glycosphingolipids were found. One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation. The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis. A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands. Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.

Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin

Abstract: Plant and animal lectins bind and cross-link certain multiantennary oligosaccharides, glycopeptides, and glycoproteins. This can lead to the formation of homogeneous cross-linked complexes, which may differ in their stoichiometry depending on the nature of the sugar receptor involved. As a precisely defined ligand, we have employed bovine asialofetuin (ASF), a glycoprotein that possesses three asparagine-linked triantennary complex carbohydrate chains with terminal LacNAc residues. In the present study, we have compared the carbohydrate cross-linking properties of two Lac-specific plant lectins, an animal lectin and a naturally occurring Lac-binding polyclonal immunoglobulin G subfraction from human serum with the ligand. Quantitative precipitation studies of the Lac-specific plant lectins, Viscum album agglutinin and Ricinus communis agglutinin, and the Lac-specific 16 kDa dimeric galectin from chicken liver demonstrate that these lectins form specific, stoichiometric cross-linked complexes with ASF. At low concentrations of ASF, 1:9 ASF/lectin (monomer) complexes formed with both plant lectins and the chicken lectin. With increasing concentrations of ASF, 1:3 ASF/lectin (monomer) complexes formed with the lectins irrespective of their source or size. The naturally occurring polyclonal antibodies, however, revealed a different cross-linking behavior. They show the formation of 1:3 ASF/antibody (per Fab moiety) cross-linked complexes at all concentrations of ASF. These studies demonstrate that Lac-specific plant and animal lectins as well as the Lac-binding immunoglobulin subfraction from specific stoichiometric cross-linked complexes with ASF. These results are discussed in terms of the structure-function properties of multivalent lectins and antibodies.

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

Abstract: N-Linked protein glycosylation in most eukaryotic cells initiates with the transfer of the oligosaccharide Glc3Man9GlcNAc2 from the lipid carrier dolichyl pyrophosphate to selected asparagine residues. In the yeast Saccharomyces cerevisiae, alg mutations which affect the assembly of the lipid-linked oligosaccharide at the membrane of the endoplasmic reticulum result in the accumulation of lipid-linked oligosaccharide intermediates and a hypoglycosylation of proteins. Exploiting the synthetic growth defect of alg mutations in combination with mutations affecting oligosaccharyl transferase activity (Stagljar et al., 1994), we have isolated the ALG6 locus. alg6 mutants accumulate lipid-linked Man9GlcNAc2, suggesting that this locus encodes an endoplasmic glucosyltransferase. Alg6p has sequence similarity to Alg8p, a protein required for glucosylation of Glc1Man9GlcNAc2.

Crystal and molecular structure of a histo-blood group antigen involved in cell adhesion: the Lewis x trisaccharide.

Abstract: This work describes the first crystal structure ever reported of a histo-blood group carbohydrate antigen: Lex. This study provides a detailed description of the conformation of two crystallographic independent molecules in a highly hydrated environment along with their hydrogen bonding properties and packing features. Some interactions observed between adjacent trisaccharides can provide the basis for involvement of Le"-Le x interactions in cellcell adhesion.

Glycosylation in lepidopteran insect cells: identification of a beta 1-->4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains

Abstract: The choice for a heterologous expression system to produce glycoprotein therapeutics highly depends on its potential to perform mammalian-like posttranslational modifications such as glycosylation. To gain more insight into the glycosylation potential of the baculovirus mediated insect cell expression system, we have studied the expression of glycosyltransferases involved in complex-type N-glycosylation. Lepidopteran insect cell lines derived from Trichoplusia ni, Spodoptera frugiperda, and Mamestra brassicae were found to express a beta 1-->4- N-acetylgalactosaminyltransferase (beta 4-GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to oligosaccharides and glycoproteins carrying a terminal beta-linked GlcNAc residue. These results suggest that Lepidopteran insect cells are capable of synthesizing complex-type carbohydrate chains containing GalNAc beta 1-->4GlcNAc (LacdiNAc) units. Baculovirus infection of the cells, however, resulted in a decrease in the activity of beta 4-GalNAcT from 80 to <1 pmol.min-1 mg-1 protein within 48 h post infection. Furthermore, considerable beta-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase activity was observed in insect cells, whether or not infected with baculovirus, as well as in the culture medium. These enzyme activities could be responsible for degradation of complex-type oligosaccharide chains containing LacdiNAc units. Our findings provide an enzymatic basis for the observation that most recombinant glycoproteins produced by baculovirus infected insect cells carry oligomannosidic-type N-linked glycans, in spite of the fact that uninfected insect cells have the potential for the synthesis of mammalian-like complex-type glycans.

Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae

Abstract: The Saccharomyces cerevisiae MNN4 gene, which is involved in mannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide, has been cloned from a lambda phage containing a yeast chromosome XI DNA fragment. The MNN4 ORF encodes a protein of 1178 amino acids. The deduced amino acid sequence shows a topology of type II membrane proteins and has a unique repeated sequence of lysine and glutamic acid at the C-terminus. Disruption and overexpression of MNN4 led to a decrease and increase, respectively, of the mannosylphosphate content in cell wall mannans prepared from both mnn4 and wild type strains. A dramatic decrease of mannosylphosphate occurs in delta mnn4 mutans. The results from genetic and biochemical experiments combine to suggest that Mnn4p is required to mediate mannosylphosphate transfer in both the core and outer chain portions of N-linked oligosaccharides.

Ganglioside GM3 inhibition of EGF receptor mediated signal transduction

Abstract: Ganglioside GM3 is a membrane component that has been described to modulate cell growth through inhibition of EGF receptor associated tyrosine kinase. In order to determine if the inhibition of cell growth by this ganglioside is specifically mediated through EGF receptor signaling, the effects of GM3 on key enzymes implicated in EGF signaling were determined and compared to another inhibitor of the EGF receptor kinase. Treatment of A IS cells in culture by GM3 or a tyrosine kinase inhibitor, leflunomide, led to the inhibition of MAP kinase and PI3 kinase activities. There was no detectable effect on phosphotyrosi ne phosphatases. In a cell free system, however, GM3 had no effect on the activity of these signaling intermediates. Leflunomide was able to directly inhibit MAP kinase activity. GM3 and leflunomide were also found to act differently on the expression of the early immediate genes. The expression of c-fos and c-jun was inhibited by both GM3 and leflunomide. The expression of c-myc, however, was only inhibited by leflunomide. These findings suggest that the action of GM3 on cell growth and signaling is specifically mediated by EGF receptor and that this ganglioside does not act directly on the intracellular intermediates of EGF receptor signaling. In addition, soluble small molecule tyrosine kinase inhibitors such as leflunomide can directly affect the activity of MAP kinases and possibly other signaling intermediates. The direct effects of leflunomide on signaling intermediates may explain the differential effects of leflunomide and GM3 on gene expression and cell growth.

CHO cells provide access to novel N-glycans and developmentally regulated glycosyltransferases.

Abstract: Chinese hamster ovary (CHO) cells express only a subset of the glycosyltransferases activities known to exist. They do not express several fucosyltransferases, galactosyltransferases, sialyltransferases or N-acetylglucosaminyltransferases. However, following mutagenesis or transfection with large amounts of DNA, rare mutants that express a transferase activity de novo have been obtained. The first CHO mutant of this type was LEC10, which expresses the N-acetylglucosaminyltransferase, GlcNAc-TIII, that adds the bisecting GlcNAc to complex N-glycans. Several analogous gain-of-function mutants have now been characterized and, all express a new glycosyltransferase activity. In several cases, expression is known to reflect gene activation at the transcriptional level. Thus, CHO cells contain quiescent glycosyltransferase genes that may be activated by mutational events. Several of these transferases have properties distinct from previously described enzymes. In fact, the most recently characterized dominant CHO mutants, LEC14 and LEC18, each express a GlcNAc-T activity that creates novel N-glycans never before observed in glycoproteins from any other source. In these and possibly other cases, it appears the CHO genome has provided access to new GlcNAc-Ts that may be difficult to identify by conventional methods.

Inhibition of glycoprotein processing by L-fructose and L-xylulose

Abstract: A number of unusual and rare carbohydrates were tested as potential inhibitors of various glycosidases, as well as inhibitors of N-linked oligosaccharide processing. The best inhibitors of several arylglycosidases and of glucosidase I were L-xylulose and L-fructose. Both of these sugars showed some inhibitory activity towards yeast alpha-glucosidase but were inactive against beta-glucosidase and other arylglycosidases. The inhibition of yeast alpha-glucosidase by L-xylulose was of a competitive nature and required a concentration of 1 x 10(-5) M for 50% inhibition. Both L-xylulose and L-fructose also inhibited the purified soybean glucosidase I, with 50% inhibition occurring at about 1 x 10(-4) M, but showed no inhibitory activity against soybean glucosidase II. When influenza virus-infected MDCK cells were raised in the presence of L-xylulose, there was a dose-dependent inhibition in the formation of complex types of oligosaccharides on the viral glycoproteins consistent with the inhibition of the processing glucosidase I. This inhibition resulted in the occurrence of oligosaccharides on the viral glycoproteins that were characterized as Glc3Man9(GlcNAc)2 structures. L-Fructose also inhibited glycoprotein processing in cell culture, and the inhibition resulted in the formation of similar oligosaccharides to those seen with L-xylulose. However, L-fructose was a poorer inhibitor than L-xylulose and required much higher concentrations for the same degree of inhibition. Neither of these compounds inhibited protein synthesis or the formation of lipid-linked saccharides in culture MDCK cells, even when tested at concentrations of 5 mg/ml (about 30 mM) of culture media.

Identification of a GDP-L-fucose:polypeptide fucosyltransferase and enzymatic addition of O-linked fucose to EGF domains

Abstract: An assay of GDP-fucose:polypeptide fucosyltransferase has been established. The enzyme catalyzes the reaction that attaches fucose through an O-glycosidic linkage to a conserved serine or threonine residue in EGF domains. The assay uses recombinant human factor VII EGF-1 domain as acceptor substrate and GDP-fucose as donor substrate. Synthetic peptides with sequences taken from five proteins previously shown to contain O-linked fucose (Harris and Spellman, 1993; Glycobiology, 3, 219-224) did not serve as efficient acceptor substrates. These synthetic peptides did not compromise complete EGF domains and did not contain all six cysteine residues that define the EGF structure. Therefore, the enzyme appears to require more than just a consensus primary sequence and likely requires that the EGF domain disulfide bonds be properly formed. The enzymatic reaction showed linear dependency of its activity on time, amount of enzyme, and substrates. Although the enzyme did not exhibit an absolute requirement for Mn2+, enzymatic activity did increase ten fold in the presence of 50 mM MnCl2. The in vitro glycosylation reaction resulted in complete conversion of the acceptor substrate to glycosylated product, and characterization of the purified product by electrospray mass spectrometry revealed that one fucose was added onto the polypeptide. Most of the enzymatic activity was found to be in the soluble fraction of CHO cell homogenates. However, when enzyme was prepared from rat liver in the presence of protease inhibitors, 37% of the activity was recovered by Triton X-100 extraction of the membrane particles after extensive aqueous washes. The result suggests that the enzyme is probably a membrane protein and, by analogy with other glycosyltransferases, probably has a 'stem' region that is very susceptible to proteolysis.

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells

Abstract: Gangliosides of the plasma membrane are important modulators of cellular functions. Previous work from our laboratory had suggested that a plasma membrane sialidase was involved in growth control and differentiation in cultured human neuroblastoma cells (SK-N-MC), but its substrates had remained obscure. We now performed sialidase specificity studies in subcellular fractions and found ganglioside GM3 desialylating activity in presence of Triton X-100 to be associated with the plasma membrane, but absent in lysosomes. This Triton-activated plasma membrane enzyme desialylated also gangliosides GD1a, GD1b, and GT1b, thereby forming GM1; cleavage of GM1 and GM2, however, was not observed. Sialidase activity towards the glycoprotein fetuin with modified C-7 sialic acids and towards 4-methylumbelliferyl neuraminate was solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in gangliosides desialylation of living cells was examined by following the fate of [3H]galactose-labelled individual gangliosides in pulse-chase experiments in absence and presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. When the plasma membrane sialidase was inhibited, radioactivity of all gangliosides chased at the same rate. In the absence of inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degraded at a considerably faster rate in confluent cultures, whereas the GM1-pool seemed to be filled by the desialylation of higher gangliosides. The results thus suggest that the plasma membrane sialidase causes selective ganglioside desialylation, and that such surface glycolipid modification triggers growth control and differentiation in human neuroblastoma cells.

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human β1,2-N-acetylglucosaminyltransferase I

Abstract: Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I.

Oxidized low density lipoprotein stimulates aortic smooth muscle cell proliferation

Abstract: We have investigated the effects of oxidized low density lipoproteins (Ox-LDL) on aortic smooth muscle cell (SMC) proliferation and the biosynthesis of glycosphingolipids. We found that Ox-LDL exerted a concentration, time, and temperature dependent alteration of cell proliferation and the biosynthesis of lactosylceramide. At low concentrations (5-10 fig/ml medium) Ox-LDL stimulated cell proliferation measured by an increase in the incorporation of pHJ-thymidine in cells and the synthesis of lactosylceramide, but not glucosylceramide synthesis. Oxidized LDL exerted a threefold increase in the incorporation of [3H]galactose and [3H]-serine in lactosylceramide. The activity of lactosylceramide synthetase; UDP-galactose glucosylceramide pi —* 4 galactosyltransferase (GaTT-2), but not glucosylceramide synthetase (GlcT-1) was stimulated by Ox-LDL. On the other hand, LDL suppressed the activity of GaIT-2 in these cells. When cells were preincubated with antibody against Ox-LDL or GaIT-2 it compromised the Ox-LDL mediated stimulation in cell proliferation and GaIT-2 activity. Similarly, D-PDMP an inhibitor of GalT2 compromised the Ox-LDL mediated effects in cells. In contrast, L-PDMP further stimulated the Ox-LDL mediated cell proliferation and GaIT-2 activity. However, preincubation of cells with preimmune rabbit serum IgG failed to abrogate Ox-LDL mediated stimulation in cell proliferation and GaIT-2 activity. In sum, we found that Ox-LDL stimulated aortic smooth muscle cell proliferation in culture. This effect resulted from Ox-LDL mediated activation of GalT-2 that produced lactosylceramide. Lactosylceramide in turn, contributed to cell proliferation. Such correlations are supportive of the notion that GaIT-2 action mediates the signal transduction of Ox-LDL contributing to cell proliferation.

Unique α2, 8-polysialylated glycoproteins in breast cancer and leukemia cells

Abstract: The N-linked oligosaccharides of neural cell adhesion molecule and the rat brain voltage-dependent sodium channel alpha subunit are specifically modified by alpha 2, 8-polysialic acid chains Until now, this carbohydrate modification has been observed only on these two proteins in mammalian cells We have identified 180-260 kDa proteins in RBL rat basophilic leukemia cells and MCF7 human breast cancer cells that are modified by alpha 2, 8-polysialylated oligosaccharides Immunofluorescence microscopy and Northern analysis confirmed that these proteins are neither the neural cell adhesion molecule nor the sodium channel alpha subunit The presence of authentic alpha 2, 8-polysialic acid on the basophilic leukemia and breast cancer proteins was confirmed by the elimination of anti-polysialic acid antibody staining after treatment with the alpha 2, 8-polysialic acid-specific endo-N-acetylneuraminidase The failure of peptide N-glycosidase F to completely remove alpha 2, 8-polysialic acid bearing oligosaccharides from the RBL protein, and the sensitivity of these oligosaccharides to beta-elimination, suggests that alpha 2, 8-polysialic acid may be found on O-linked oligosaccharides This identification of new alpha 2, 8-polysialylated proteins in RBL basophilic leukemia and MCF7 breast cancer cells suggests that alpha 2, 8-polysialylation of glycoproteins may be more widespread than originally believed, especially in cancer cells

A galectin links the aggregation factor to cells in the sponge (Geodia cydonium) system

Abstract: The cDNA for the full-length lectin from the marine sponge Geodia cydonium was cloned. Analysis of the deduced aa sequence revealed that this lectin belongs to the group of galectins. The full-length galectin, which was obtained also in a recombinant form, has an M(r) of 20,877; in the processed form it is a 15 kDa polypeptide. The enriched aggregation factor from G.cydonium also was determined to contain, besides minimal amounts of the galectin, a 140 kDa polypeptide which is involved in cell-cell adhesion. Monoclonal antibodies have been raised against this protein; Fab' fragments prepared from them abolished cell-cell reaggregation. Cell reaggregation experiments revealed that the aggregation factor is an essential component in the aggregation process but it requires likewise the presence of the galectin. Antibodies against the galectin blocked the aggregation factor-mediated cell adhesion. A plasma membrane component was identified (a 29 kDa polypeptide) which interacted with the aggregation factor in the presence of galectin; binding could be blocked both by antibodies against the galectin as well as against the aggregation factor. Immunohistochemical analysis revealed that spherulous cells contain the galectin but not the aggregation factor. By laser scanning microscopy, it is shown that both the aggregation factor and the galectin are located at the rim of the cells. From these data we conclude that the G.cydonium aggregation factor binds to the cells via a galectin bridge.

Transcription of the β-galactoside α2,6-sialyltransferase gene in B lymphocytes is directed by a separate and distinct promoter†

Abstract: A single human gene, SIAT1, encodes the beta-galactoside alpha 2,6-sialyltransferase from which multiple mRNA isoforms are generated. In rat, expression of the hepatic mRNA isoform (Form 1) has been defined with respect to the transcriptional initiation site and promoter region. We show here that a similar hepatic SIAT1 mRNA isoform exists in human. Another human mRNA isoform, a mature B-cell-specific mRNA isoform (Form 2), was previously reported. Here, we used 5'-RACE and S1 nuclease protection analysis to define the 5'-untranslated region of Form 2 human SIAT1 mRNA. We demonstrate conclusively that Form 2 mRNA is initiated from a point completely distinct from that of Form 1 mRNA. A number of cis-acting regulatory elements residing immediately 5'of the Form 2 initiation site includes AP-1, AP-2, NF-kappa B, NF-IL6, C/EBP, and CREB. A TATAA box is also present 29 bp 5' of the transcriptional initiation site. CAT reporter gene expression from serially-truncated segments of the 5'-flanking region of the Form 2 initiation site indicates that the segment between -784 and +125 was sufficient to promote high level CAT expression in Louckes, a mature B-cell line. The 5'-flanking region to the human Form 1 initiation site is competent in expression of CAT upon transfection of the fusion construct into HepG2, a human hepatoma cell line. Cellular specificity of expression is apparently retained. Louckes cells expressed CAT efficiently from Form 2 promoter but only marginally from the Form 1 promoter. In contrast, CAT expression from Form 1 promoter is more efficient than from the Form 2 promoter in HepG2 cells.

Complexity in O-linked oligosaccharide biosynthesis engendered by multiple polypeptide N-acetylgalactosaminyltransferases.

Abstract: Linkage of JV-acetylgalactosamine (GalNAc) to the hydroxyl group of serine and threonine in a peptide context initiates the production of vertebrate O-glycosylation, although the identity, specificity and location of components involved in this posttranslational modification have been debated. Studies by Robert Hill and others have found that O-glycosylation begins in one or more compartments of the Golgi apparatus (Hanover et al, 1982; Elhammer and Kornfeld, 1984; Roth, 1984; Abeijon and Hirschberg, 1987; Deschuyteneer et al, 1988; Piller et al, 1990; Roth et al, 1994; Schweizer et al, 1994). While various cell types were used, others have indicated that the endoplasmic reticulum is the site of O-glycari biosynthesis (Strous, 1979; Cummings et al, 1983; Patzelt and Weber, 1986; Perez-Vilar et al, 1991; Ellinger and Pavelka, 1992). Although it is possible that a significant number of laboratories are in error, alternatively, all may be correct with these differing results due to a source of complexity in O-glycan biosynthesis not previously defined.

Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus.

Abstract: The presence of chitinase activity in human serum has recently been described by us On that occasion we speculated on the possible role of mammalian chitinases as a defense mechanism against chitin-containing pathogens The results of the present study substantiate our hypothesis We demonstrate and partially characterize the chitinase activities that are present in plasma of guinea pigs and in homogenates of Afumigatus with the aid of the substrates MU-[GlcNAc]2,3 and also with glycol [3H]chitin Upon infection with Afumigatus the serum chitinase activity levels in the circulation of pathogen-free guinea pigs increased in a time-dependent manner The increase was also dependent on the size of the infecting fungal inoculum Antifungal treatment diminished the increases The increased chitinase activity was of guinea pig origin The activity of beta-hexosaminidase showed a very slight increase subsequent to the infection The activities of three other enzymes of lysosomal origin (alpha-mannosidase, beta-galactosidase and beta-glucosidase) did not increase

Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications

Abstract: Mannose-binding proteins (MBPs), members of the collectin family, have been implicated as lectin opsonins for various viruses and bacteria. Two distinct but related MBPs, MBP-A and MBP-C, with approximately 55% identity at the amino acid level, have been previously characterized from rodents. In humans, however, only one form of MBP has been characterized. In this paper we report studies elucidating the evolution of primate MBPs. ELISA and Western blot analyses indicated that rhesus and cynomolgus monkeys have two forms of MBP in their sera, while chimpanzees have only one form, similar to humans. Two distinct MBP cDNA clones were isolated and characterized from a rhesus monkey liver cDNA library. Rhesus MBP-A is closely related to the mouse and rat MBP-A, showing 77% and 75% identity at the amino acid level, respectively. Rhesus MBP-A also has three cysteines at the N-terminus, similar to mouse and rat MBP-A and human MBP. Rhesus MBP-C shares 90% identity with the human MBP at the amino acid level and has three cysteines at the N-terminus, in contrast to two cysteine residues found in rodent MBP-C. A stretch of nine amino acids close to the N-terminus, absent in both mouse and rat MBP-A, but present in rodent MBP-C, chicken and human MBPs, is also found in the rhesus MBP-A. The phylogenetic analysis of rhesus and other mammalian MBPs, coupled with the serological data suggest that at least two distinct MBP genes existed prior to mammalian radiation and the hominoid ancestor apparently lost one of these genes or failed to express it.

Oral estrogen treatment induces a decrease in expression of sialyl Lewis x on α1 glycoprotein in females and male-to-female transsexuals

Abstract: The effect of estrogen on the glycosylation of alpha 1-acid glycoprotein was studied in women using oral contraceptives and in male-to-female transsexuals receiving oral or transdermal estrogen treatment. Oral estrogen treatment induced an increase in degree of branching and a decrease in fucosylation and sialyl Lewis x expression on alpha 1-acid glycoprotein compared to individuals receiving no estrogens or transdermal estrogen treatment. The effect on the glycosylation of alpha 1-acid glycoprotein was less in the male-to-female transsexuals receiving oral estrogens in combination with cyproterone acetate, a blocker of the androgen receptor with progestagen-like effects. This was of comparable magnitude as in women using oral contraceptives containing both estrogen and progestagen. We conclude that oral estrogens have an identical effect on the hepatic glycosylation of alpha 1-acid glycoprotein in both males and females and that progestagen reduces this effect. These results show that oral estrogens induce an effect on the glycosylation of alpha 1-acid glycoprotein opposite to that induced by inflammation. Oral estrogens can therefore modulate the glycosylation dependent inflammatory actions of alpha 1-acid glycoprotein, while this is not the case with transdermal estrogens. In all likelihood, estrogen receptors on the hepatocyte must play a significant role in the observed effect.

The hypothetical N-glycan charge: a number that characterizes protein glycosylation.

Abstract: The production of recombinant glycoprotein therapeutics requires characterization of glycosylation with respect to the lot-to-lot consistency. Here we introduce the ?hypothetical N-glycan charge Z' as a parameter that allows to characterize the protein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein is deduced from the N-glycan mapping profile obtained via HPAE-PAD. In HPAEC, N-glycans are clearly separated according to their charge, i.e., their number of sialic acid residues, providing distinct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined as the sum of the products of the respective areas (A) in the asialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region, each multiplied by the corresponding charge: [formula: see text] Thus, a glycoprotein with mostly C4-4* structures will provide Z approximately equal to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3* structures will amount to Z approximately equal to 300 (e.g., bovine fetuin, Z = 290), a glycoprotein with mostly C2-2* structures will have Z approximately equal to 200 (e.g., human serum transferrin, Z = 207, or human plasma AT III, Z = 180), and a glycoprotein carrying only high-mannose type or trunkated structures will provide Z approximately equal to 0 (e.g., bovine pancreas ribonuclease B, Z = 15, and hen ovomucoid, Z = 15, respectively). The determination of Z was validated in multiple repetitive experiments and proved to be highly accurate and reliable. Z may therefore be regarded as a new and characteristic parameter for protein N-glycosylation.