A family of novel, acidic N-glycans in Bowes melanoma tissue plasminogen activator have L2/HNK-1-bearing antennae, many with sulfation of the fucosylated chitobiose core.

A family of about 20 novel acidic bi- and tri-antennary N-glycans, amounting to almost half those expressed on Bowes melanoma tissue-plasminogen activator (t-PA) were found to possess Galbeta1-->4GlcNAcbeta1-->, sulfated and sialylated GalNAcbeta1-->4GlcNAcbeta1--> or sulfated GlcAbeta1--> 3Galbeta1-->4GlcNAcbeta1--> antennae, of which those containing sulfated GlcA, depicting the L2/HNK-1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK-1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6-branched antenna and an alpha2-->6 sialylated GalNAc on the other. The N-glycosylation sequon containing Asn448, which is known to express all of the sulfate-carrying N-glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core-sulfate group of the N-glycan is proposed to reduce mobility of the carbohydrate in the region of the t-PA active site. Because of the 'brain-type' nature of the N-glycans described in this neuro-ectodermal cell line, the possibility of neural t-PA interacting with the L2/HNK-1-recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed.

Glycosylation of a CNS-specific extracellular matrix glycoprotein, tenascin-R, is dominated by O-linked sialylated glycans and "brain-type" neutral N-glycans.

As a member of the tenascin family of extracellular matrix glycoproteins, tenascin-R is located exclusively in the CNS. It is believed to play a role in myelination and axonal stabilization and, through repulsive properties, may contribute to the lack of regeneration of CNS axons following damage. The contrary functions of the tenascins have been localized to the different structural domains of the protein. However, little is known concerning the influence of the carbohydrate conjugated to the many potential sites for N - and O -glycosylation (10-20% by weight). As a first analytical requirement, we show that >80% of the N -glycans in tenascin-R are neutral and dominated by complex biantennary structures. These display the "brain-type" characteristics of outer-arm- and core-fucosylation, a bisecting N -acetylglucosamine and, significantly, an abundance of antennae truncation. In some structures, truncation resulted in only a single mannose residue remaining on the 3-arm, a particularly unusual consequence of the N -glycan processing pathway. In contrast to brain tissue, hybrid and oligomannosidic N -glycans were either absent or in low abundance. A high relative abundance of O -linked sialylated glycans was found. This was associated with a significant potential for O -linked glycosylation sites and multivalent display of the sialic acid residues. These O -glycans were dominated by the disialylated structure, NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc. The possibility that these O -glycans enable tenascin-R to interact in the CNS either with the myelin associated glycoprotein or with sialoadhesin on activated microglia is discussed.

Sialylated N-glycans in adult rat brain tissue--a widespread distribution of disialylated antennae in complex and hybrid structures.

This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40% of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7%, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for 'brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core alpha1-6-linked and outer-arm alpha1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewis(x) and NeuAc alpha2-3 substituted Galbeta1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal beta1-3-linked galactose residues was observed. The presence of beta1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAc alpha2-3Galbeta1-3(NeuAc alpha2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one beta1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing beta1-3-linked galactose and 'polylactosamine' N-glycans with one to three terminal beta1-3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal beta1-3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.

Identification of lectin-purified neural glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA receptor subunits: conservation of glycosylation at the synapse.

The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for approximately 50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.

Neutral N-glycans in adult rat brain tissue--complete characterisation reveals fucosylated hybrid and complex structures.

Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man(9-5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)-branched triantennary glycans (1%) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm alpha(1,3)-fucosylation (forming the Lewis[x] determinant), alpha(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man alpha(1,3)-arm. This isomeric form is indicative of the action of a novel beta-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.

Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland.

The glycosylation of a number of constituents of human saliva is known to modify its biological roles, such as its lubricating properties and binding of microbial flora. Gillece-Castro et al. [Gillece-Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17358-17368] have proposed that the major glycan on the salivary proline-rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non-fucosylated antenna mediated adherence to a peridontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co-exist in the parotid gland are not fully known. In view of the influence of outer-arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix-assisted laser-desorption/ionisation (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed-phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal-phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2-aminobenzamide-labelled arabinose oligomers instead of the corresponding oligomers from partially hydrolysed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were additionally found substituted with sialic acid (both alpha2-3-linked and alpha2-6-linked) and sulphate. These glycans were mainly bi- and tri-antennary sugars with up to five and seven fucose residues respectively, containing fucose alpha1-3-linked to the outer-arm GlcNAc residues and alpha1-2-linked to the galactose. All fucosylated structures contained a core (alpha1-6-linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amounts of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addition of extra sialic acid moieties. The possible functional significance of highly fucosylated N-glycans is discussed in relation to their modification of the availability of other non-reducing terminal monosaccharides for recognition processes.

The glycosylation of Bowes melanoma tissue plasminogen activator: lectin mapping, reaction with anti-L2/HNK-1 antibodies and the presence of sulphated/glucuronic acid containing glycans.

The glycosylation of tissue plasminogen activator (t-PA) obtained from the Bowes melanoma cell line was re-examined using methods of serial lectin affinity chromatography coupled with Bio-Gel P-4 gel filtration chromatography and exoglycosidase sequencing. This study clarified an earlier discrepancy in the literature and confirmed that the major complex N-linked glycans on Bowes t-PA that carry sialic acid as their sole charged group are bi-antennary, core fucosylated, with terminal N-acetylgalactosamine residues. We also report the characterization of a series of related and previously unidentified sialylated glycans. Further we show that Bowes t-PA expresses glucuronic acid/sulphate containing N-linked glycans and is recognized by anti-carbohydrate L2/HNK-1 monoclonal antibodies. The presence on Bowes t-PA of glycans associated primarily with the nervous system is consistent with its expression in a cell line of neuroectodermal origin.

Glycosylation in parasitic protozoa of the trypanosomatidae family.

Over the last few years enormous interest has been shown in the structures of the glycan moieties of various parasite surface glycoconjugates. Structures have been determined for the glyco-components of glycosylphosphatidylinositol (GPI) protein membrane anchors, for asparagine-linked oligosaccharides, and for the glycans of complex glycolipids. The following attempts to illustrate a few of the most salient observations with regard to the structures and possible functions of parasite surface glycans.

Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins.

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.

Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins.

The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.

Primary sequence dependence of conformation in oligomannose oligosaccharides.

The oligomannose series of oligosaccharides from bovine thyroglobulin (BTG) and the variant surface glycoprotein (VSG) of Trypanosoma brucei have been isolated and sequenced by 1H NMR. The structure of Man9GlcNAc2, the parent molecule of the series, is shown below. Structural isomerism occurs within this series through the removal of residues D1, D2, D3, and C. Using spin-spin coupling and chemical shift data the rotamer distributions about the dihedral angle omega for the Man alpha 1-6Man beta and Man alpha 1-6Man alpha linkages were determined for each member of the series. It is shown that the dihedral angle omega of the Man alpha 1-6Man beta linkage exhibits low flexibility with a preference for the omega = 180 degrees conformation when residue D2 is present and high flexibility when this residue is absent. Flexibility of omega for the Man alpha 1-6Man alpha is largely independent of primary sequence and is intermediate between the two Man alpha 1-6Man beta extremes, again with a preference for the omega = 180 degrees conformation. [see text] There are, however, data which indicate that removal of residue D3 may confer additional flexibility upon the dihedral angle omega of the Man alpha 1-6Man alpha linkage. Molecular graphics modelling, together with chemical and enzymatic modification studies, suggest that the origin of the observed primary sequence dependence of the Man alpha 1-6Man beta linkage arises from steric factors. On the basis of these observations taken together with previous work, it is postulated that recognition of individual oligomannose conformations may play a role in the control of N-linked oligosaccharide biosynthesis.

Common organization of chromosomal loci for production of different capsular polysaccharides in Haemophilus influenzae.

Cloned Haemophilus influenzae type b capsulation genes were used as hybridization probes to isolate DNA from the capsulation loci (cap) of other serotypes of H. influenzae. Mapping of the resulting clones and Southern hybridization analysis of chromosomal DNAs from type a, b, c, and d strains showed that in each strain cap was organized in the same way: a central DNA segment specific to each serotype flanked by DNA segments of common structure. We infer that enzymes necessary for the synthesis of specific capsular polysaccharide are encoded in the central segment of cap, while proteins involved in a more general way in the process of capsulation are encoded in the flanking segments. Studies of the function of the DNA in one of these non-serotype-specific flanking segments (J. S. Kroll, I. Hopkins, and E. R. Moxon, Cell 53:347-356, 1988) have previously identified a gene encoding a protein necessary for polysaccharide export, an event now deduced to proceed by a mechanism independent of the nature of the disaccharide subunit in the polysaccharide. The near-total duplication of cap that has been found in most type b strains was not found at the analogous locus in the other serotypes. This reinforces our previous hypothesis, based on study of type b strains alone, that while such a duplication is unnecessary for capsulation, it confers some unexplained survival advantage on the widely prevalent strains with this clinically important serotype.

Characterization of the cross-reacting determinant (CRD) of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein.

The cross-reacting determinant (CRD epitope) of the glycosyl-phosphatidylinositol (GPI) membrane anchor of Trypanosoma brucei variant surface glycoprotein has been analysed by selective chemical and enzymic modification of the isolated GPI structure combined with the use of a competitive ELISA inhibition assay for the detection of CRD epitopes. The data show that the CRD consists of at least three overlapping epitopes involving different regions of the molecule including the inositol 1,2-cyclic phosphate, the non-N-acetylated-glucosamine residue and the galactose branch. Although the presence of all three of these structural features is required for quantitative binding of anti-CRD antibodies in ELISA and Western blotting, the Western blot reaction obtained in the presence of any one epitope is still significant. The use of anti-CRD antibodies for the detection of GPI anchors is discussed.

Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.

Composition of the lipopolysaccharide from different capsular serotype strains of Haemophilus influenzae.

Lipopolysaccharide (LPS) from all six serotype strains of Haemophilus influenzae was similar in composition. The oligosaccharide, of each LPS, was composed of glucose, galactose, heptose and 2-keto-3-deoxyoctonic acid. The lipid A was composed of glucosamine, phosphate and the fatty acids 14:0 and 3-OH 14:0. Each LPS also contained ethanolamine and ethanolamine phosphate, and the oligosaccharides from two strains additionally contained small amounts of glucosamine. Although the LPS was similar in composition, different serotypes had quantitative differences, especially in the galactose content, which correlated with the antigenic specificity of their homologous antisera and with their mobility on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A survey by SDS-PAGE showed that LPS from strains of the serotypes a, c and d was characteristically of lower Mr than the LPS from most (80%) serotype b strains.

Structure of the sidechain of lipopolysaccharide from Pseudomonas syringae pv. morsprunorum C28.

The sidechain of the lipopolysaccharide from the phytopathogen Pseudomonas syringae pv. morsprunorum C28 was shown to be composed of D-rhamnose. Using 1H and 13C-NMR spectroscopy, methylation analysis, Smith degradation and optical rotation data, the repeat unit was found to have the structure: ----3)-D-Rhap-(alpha 1----3)-D-Rhap-(alpha 1----2)-D-Rhap-(alpha 1---- and a degree of polymerization of approximately 70. Attention is drawn to the possible prevalence of D-6-deoxyhexoses in the lipopolysaccharides of plant pathogenic bacteria.

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination.

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.