Biomedical subjects

A Dell

Publications and source records attributed to A Dell.

At least 109 records · Page 6Linked to original sources

PubMed1994

Glycans as targets for monoclonal antibodies that protect rats against Trichinella spiralis.

We have investigated the role of glycans on Trichinella spiralis antigens in recognition by rat monoclonal antibodies (mAbs) which protect rat pups against challenge with the parasite. In pups born to infected dams or pups passively immunized with mAbs, antibodies eliminate a challenge dose from the intestine within hours ('rapid expulsion'). Because such dramatic protection can be afforded by mAbs, we have sought to characterize the parasite antigens they target. In this report we show that protective antibodies were unable to bind excretory/secretory (ES) antigens deglycosylated with trifluoromethanesulphonic acid (TFMS). In addition, oligosaccharides isolated from glycoproteins by alkaline hydrolysis or peptide: N glycosidase F (PNGase F) digestion were bound by protective, but not non-protective, mAbs. Glycans affinity purified with protective mAb 9D bound to all but one protective mAb. These antibodies have been shown previously to bind to the surfaces of intact larvae, indicating that the glycan is exposed on the parasite surface. Candidate glycans that may be involved in binding protective mAbs have unusual tri- and tetra-antennary structures with terminal tyvelose moieties (Reason et al., Glycobiology, 4, 000-000, 1994). Coating of the larval surface with such glycans may serve to protect the parasite and its secreted products from enzymatic attack as the parasite travels to and resides in its epithelial niche.

Animals↗

PubMed1994

Novel tyvelose-containing tri- and tetra-antennary N-glycans in the immunodominant antigens of the intracellular parasite Trichinella spiralis.

The larval stage of the intestinal nematode, Trichinella spiralis, secretes and displays on its cuticle a number of antigenically cross-reactive glycoproteins. These so-called TSL-1 antigens induce a powerful antibody response in parasitized animals. In rats, anti-TSL-1 antibodies mediate a protective immunity that expels invading larvae from the intestine. The vast majority of anti-TSL-1 antibodies are specific for glycans. Although the biological functions of TSL-1 antigens are not known, the powerful effect of glycan-specific antibodies on the intestinal survival of T. spiralis suggests that they play an important role in parasite establishment. Little is known about the structures of the glycans present on the TSL-1 glycoproteins. Recent studies have suggested, however, that the antigens contain very unusual glycans (Wisnewski, N., McNeil, M., Grieve, R.B. and Wassom, D.L., Mol. Biochem. Parasitol., 61, 25-36, 1993). Sugar and linkage analysis of the combined secreted products unexpectedly showed that a major terminal sugar is tyvelose (3,6-dideoxy-D-arabino-hexose; Tyv) which has previously been found only in certain gram-negative bacterial lipopolysaccharides. In this paper, we report the first rigorous structural study of oligosaccharides released from TSL-1 antigens by peptide N-glycosidase F digestion. Using strategies based on fast atom bombardment mass spectrometry (FAB-MS), we have discovered a novel family of tri- and tetra-antennary N-glycans whose antennae are comprised of the tyvelose-capped structure: Tyv1,3GalNAc beta 1,4(Fuc alpha 1,3)GlcNAc beta 1-. Thus a major population of TSL-1 glycans contains clusters of hydrophobic terminal structures which are likely to be highly immunogenic.

Animals↗

PubMed1993

Trehalose-containing lipooligosaccharides of Mycobacterium gordonae: presence of a mono-O-methyltetra-O-acyltrehalose "core" and branching in the oligosaccharide backbone.

Past evidence has indicated that Mycobacterium gordonae, as isolated from soil and as an occasional opportunistic pathogen, exists as a serocomplex. We now demonstrate that the basis of seroreactivity and diversity is a novel series of alkali-labile, trehalose-containing lipooligosaccharides (LOS). The structures from two strains were established by per-O-methylation, partial acid hydrolysis, infrared and high-field NMR spectroscopy, electron-impact MS, and fast atom bombardment/mass spectrometry of the native lipooligosaccharides and hydrolysis products. The structure of the major lipooligosaccharide, LOS-I, of M. gordonae strain 989 was defined as 2-O-CH3-4-O-CH3CO-alpha-L-Fucp-(1-->3)- beta-D-Glcp-(1-->3)-2-O-CH3-alpha-L-Rhap-(1-->3)-[beta-D-Xylp++ +-(1-->2)-]- alpha-L-Rhap-(1-->3)-beta-D-Glcp-(1-->3)-alpha-L-Rhap-(1-->3 )-6-O- CH3-alpha-D-Glcp-(1<-->1)-2,3,4,6-tetra-O-acyl-alpha-D-Glcp, which was further glycosylated at C-3 of the terminal 2-O-CH3-4-O-CH3CO-alpha-L-Fucp by an incompletely defined N-acyl derivative of 4-amino-4,6-dideoxy-2,3-di-O-CH3-Galp. The structure of the major lipooligosaccharide, LOS-I, of a second strain of M. gordonae (strain 990) was defined as alpha-L-Rhap-(1-->2)-3-O-CH3-alpha-L-Rhap- (1-->3)-[beta-D-Xylp-(1-->2)-]-alpha-L-Rhap-(1-->3)-beta-D-G lcp- (1-->3)[beta-D-Xylp-(1-->2)-]-alpha-L-Rhap- (1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-L-Rhap-(1--> 3)-6-O-CH3- alpha-D-Glcp-(1<-->1)-2,3,4,6-tetra-O-acyl-alpha-D-Glcp. The other minor LOSs from both strains were also defined. Both families of LOSs from the two strains contain a novel mono-6'-O-CH3-2,3,4,6-tetra-O-acyltrehalose unit, representing the first example of such a unit among the LOSs isolated to date from mycobacteria. Also, the more polar antigenic products, LOS-I, -II', -II", and -III from M. gordonae 989 and LOS-I, -II, and -II' from M. gordonae 990, are characterized by branching of the oligosaccharide backbone, the first instance of sugar branching in these products. In the case of LOS-I and -III from M. gordonae 989, the branch consists of a terminal (t)-beta-D-Xylp unit, whereas in LOS-II' and -II", they are (t)-3-O-CH3-beta-D-Xylp and (t)-alpha-D-Araf, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Acylation↗

PubMed1993

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides.

Derivatisation/FABMS strategies applicable to the structure analysis of low microgram quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitrous acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.

Acetylation↗

PubMed1993

Studies on the carbohydrate moiety and on the biosynthesis of rat C-reactive protein.

Rat C-reactive protein (CRP) is a pentameric glycoprotein composed of five apparently identical monomers, two of which form a disulfide-linked dimer (Rasosouli, M., Sambasivam, H., Azadi, P., Dell, A., Morris, H. R., Nagpurkar, A., Mookerjea, S., and Murray, R. K. (1992) J. Biol. Chem. 267, 2947-2954). In this study, the nature of the oligosaccharide chain of rat CRP was investigated by fast atom bombardment-mass spectrometry (FAB-MS), and general features of its biosynthetic pathway were also analyzed. FAB-MS, electrospray-mass spectrometry, and linkage analysis demonstrated that each monomer of rat CRP contained one oligosaccharide chain, predominantly a disialylated biantennary structure, attached to Asn-128. The biosynthesis of rat CRP was studied by immunoprecipitation of CRP synthesized in vitro and by cultured hepatocytes. The results revealed that each monomer of rat CRP was synthesized individually as a single-chain precursor with a cleavable signal sequence. The translocated species was sensitive to digestion by endoglycosidase H (endo H), indicating that it possessed a high mannose oligosaccharide. Rat CRP acquired the ability to bind to phosphorylcholine-Sepharose and to form the dimeric and oligomeric species prior to acquiring resistance to endo H. Studies using tunicamycin revealed that the N-linked oligosaccharide present in rat CRP was not required for formation of its dimeric component, oligomerization, ability to bind to phosphorylcholine, or secretion. The non-glycosylated rat CRP, however, was still able to bind to phosphorylcholine-Sepharose and to be secreted by hepatocytes.

Animals↗

PubMed1993

Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin.

Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.

Animals↗

PubMed1993

The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s).

A new class of inositol phosphates containing energy-rich pyrophosphoryl residues has been characterized. D/L-1-Diphosphoinositol pentakisphosphate(s) and D/L-bis-(1,4)-diphosphoinositol tetrakisphosphate(s) are present as soluble ionic species in the cytosol of amoebae (Dictyostelium discoideum) at concentrations in the range of 0.05-0.25 mM. These compounds are rapidly metabolized in intact cells and can be synthesized in cell lysates from myo-inositol hexakisphosphate in the presence of ATP. Their phosphomonoester groups have predicted C-O-P bond energies of between 3.3 and 4 kcal mol-1; however, the bond energies of the P-O-P links in their diphosphate moieties are 6.6 kcal mol-1 and hence similar to the equivalent bonds in ADP, indicating a potential role for these compounds as phosphate donors in phosphotransferase reactions. Compounds with similar chromatographic properties are found in a variety of mammalian cell types.

Animals↗

PubMed1993

Structural studies of a novel type of tetraantennary sialoglycan unit in a carbohydrate-rich glycopeptide isolated from the fertilized eggs of Indian Medaka fish, Oryzias melastigma.

A novel carbohydrate-rich sialoglycopolyprotein of apparent molecular mass approximately 7000 Da was isolated from the fertilized eggs of the Medaka fish species, Oryzias melastigma. The glycoprotein was identified as a member of the L-hyosophorin family because it exhibited the following several distinctive features of L-hyosophorin molecules: (a) it contains a high proportion of carbohydrate (90% by weight), and (b) the amino acid sequence of the apopeptide was identical with that of the Oryzias latipes L-hyosophorin which has previously been demonstrated to be derived from a high molecular weight form of hyosophorin, i.e. H-hyosophorin, present in the cortical vesicles of unfertilized eggs. The apoprotein of H-hyosophorin is composed of tandem repeats of the L-hyosophorin apopeptide, i.e. it is a polyprotein. The structure of the carbohydrate portion of purified L-hyosophorin of O. melastigma was studied by composition and methylation analysis, selective chemical (periodate-Smith degradation; hydrazinolysis-nitrous acid deamination), and enzymatic (endo-beta-galactosidase; peptide:N-glycanase) degradation, together with instrumental methods (fast atom bombardment-mass spectrometry and 1H NMR). O. melastigma L-hyosophorin was found to contain two types of large, branched tetraantennary glycan units capped with sialic acids. The two glycans differ with respect to the branching pattern of the trimannosyl core (x = 4 or 6 in Eq. A). [formula: see text] The possible physiological significance of the hyosophorin family is discussed in the light of their unique structural features.

Animals↗

PubMed1993

Further structural definition of a new family of glycopeptidolipids from Mycobacterium xenopi.

The highly antigenic surface glycolipids of serovars of the Mycobacterium avium complex are glycopeptidolipids of the general structure [formula: see text] However, it has recently been shown [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063; Rivière, M., & Puzo, G. (1992) Biochemistry 31, 3575-3580] that the characteristic glycopeptidolipid of a strain of Mycobacterium xenopi is of the structure [formula: see text] [monosaccharide, 3-O-CH3-6-deoxy-alpha-L-talopyranose; tetrasaccharide, 2,3,4-tri-O-CH3-alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L- rhamnopyranose(1-->3)2,4-di-O-acetyl/lauryl-6-deoxy-alpha-L-glucopyranos e]. The type of analyses previously applied to the glycopeptidolipids of M. avium combined with fast atom bombardment mass spectrometry allowed the recognition of other variants of this structure in other strains of M. xenopi: [formula: see text] in which the monosaccharide is 2-O-acetyl-3-O-CH3-6-deoxy-alpha-L-talopyranose or 3-O-CH3-6-deoxy-alpha-L-talopyranose, and the disaccharide is 4-O-octanoyl(or decanoyl)-alpha-L- rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose or alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose. Recognition of serine-containing glycopeptidolipids substituted in one case with a tetrasaccharide and in another case with a disaccharide unit implies that M. xenopi, an important opportunistic pathogen, exists as a serocomplex in nature.

Amino Acid Sequence↗

PubMed1993

Carbohydrate analysis.

Carbohydrate analysis is an active field that is expanding rapidly. Hundreds of new structures are reported each year and methods for screening glycopolymers for known structures are now becoming accessible to the nonspecialist. Detailed structure analysis of recombinant glycoproteins has become relatively routine in specialist laboratories. Rapid advances are being made in the understanding of structure and function of biologically active carbohydrates that are of interest to the pharmaceutical industry.

Animals↗

PubMed1993

The Lewis x epitope is a major non-reducing structure in the sulphated N-glycans attached to Asn-65 of bovine pro-opiomelanocortin.

The N-terminal glycopeptide of pro-opiomelanocortin (POMC), designated as the 16K fragment, is highly conserved throughout vertebrates from amphibians to mammals and is likely therefore to have an important functional role. In this paper, we report the first structural characterization of N-glycans attached to asparagine-65 of a 16K glycopeptide. The 16K fragment was isolated from bovine pituitaries and the N-glycans were analysed using fast atom bombardment mass spectrometry together with sugar and linkage analysis. Sulphated-N-acetylgalactosamine-capped antennae, typical of the pituitary glycohormones, were present in the major acidic components. The POMC oligosaccharides are distinct from those of the pituitary glycohormones because the sulphate is exclusively located on the 3-arm of biantennary structures and, in addition, a significant proportion of the molecules carry the Lewis x epitope. It is probable that these differences reflect the absence of a tripeptide motif in POMC which fully conforms to the criteria previously defined for the recognition sequence for the N-acetylgalactosamine transferase that is specific for the pituitary glycohormones [Smith and Baenziger (1992) Proc. Natl. Acad. Sci. USA, 89, 329-333]. It remains to be seen whether the Lewis x epitope is involved in selectin-mediated events, but previous studies suggest that the sulphated moieties are unlikely to play a major role in clearance. The Lewis x epitope is also present in the neutral N-linked oligosaccharides, together with a variety of other antennae including a rarely found fucosylated GalNAc-GlcNAc structure.

Acetylgalactosamine↗

PubMed1993

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid.

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.

Amino Acid Sequence↗

PubMed1993

Structural definition of the non-reducing termini of mannose-capped LAM from Mycobacterium tuberculosis through selective enzymatic degradation and fast atom bombardment-mass spectrometry.

The application of extracellular arabinases from a Cellulomonas sp. and fast atom bombardment-mass spectrometry (FAB-MS) provided new insight into the structure of lipoarabinomannan (LAM) of Mycobacterium tuberculosis, a key molecule in the pathogenesis and physiology of the tubercle bacillus. Previously, the non-reducing arabinan ends of LAM from the virulent (Erdman) strain of M. tuberculosis were shown to be 'capped' by short (alpha 1-->2)-linked mannopyranose (Manp)-containing oligosaccharides, a product called ManLAM. The structural relationship between these Manp units and the underlying arabinofuranose (Araf)-containing arabinan was examined by digesting ManLAM from M.tuberculosis Erdman with the Cellulomonas enzyme, resolving fragments by various means and subjecting the derivatized oligoglycosylalditols to FAB-MS. The sequences Manp2Araf4, Manp3Araf4 and Manp1-6Araf6 were recognized as the major terminal motifs. Upon complete structural definition, all of the Ara6-containing products were shown to be based on a 3,5-linked branched Araf unit, whereas those containing Ara4 were linear. Minor non-mannosylated terminal arrangements containing Ara4-6, branched, linear and cyclical, were also recognized. In addition, the mannan 'core' of ManLAM was isolated from enzyme digests and shown to contain segments of the phosphatidylinositol anchor and a 'stub' of the arabinan side-chain in the form of a 'linker' alpha-Araf-(1-->5)-Araf unit attached to C-2, apparently of the penultimate 2,6-linked Manp residue. The structural unravelling of this complex molecule further substantiates the case for structural and biological similarities to the enterobacterial lipopolysaccharides/lipoglycans and other important 'capped' lipooligomers such as the lipooligosaccharides of Neisseria species and the lipophosphoglycan of Leishmania promastigotes.

Carbohydrate Sequence↗

PubMed1992

Localization of O-GlcNAc modification on the serum response transcription factor.

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide, tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and 374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion, together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions. The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant from the major sites of phosphorylation catalyzed by casein kinase II.

Acetylglucosamine↗

PubMed1992

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.

Acetyl Coenzyme A↗

PubMed1992

Determination of the structure of the capsular antigen of Escherichia coli O8:K46:H30, using FABMS and 2D-NMR spectroscopy.

The structure of the capsular antigen from Escherichia coli O8:K46:H30 was elucidated by methylation analysis and 1D and 2D 1H- and 13C-NMR spectroscopy, and by methylation analysis, 1D- and 2D-NMR spectroscopy, and FABMS of the oligosaccharide-alditol obtained after dephosphorylation of the polymer with aqueous hydrofluoric acid. The capsular polymer is of the teichoic acid type and has the following repeating unit. [Formula: see text]

Antigens, Bacterial↗

PubMed1992

Stored dolichyl pyrophosphoryl oligosaccharides in Batten disease.

Each of the 3 childhood forms of Batten disease, juvenile (JB), late-infantile (LIB), and infantile (IB), have abnormally high brain concentrations of dolichyl pyrophosphoryl oligosaccharides (Dol-PP-OS). In this study, the carbohydrate portions of Dol-PP-OS were analysed: in JB and LIB, they range in size from Man2GlcNAc2 to Glc3Man9GlcNAc2, predominant components being Man5-7GlcNAc2 and Glc3Man7GlcNAc2. In IB, they range from Man6-9GlcNAc2, no glucose containing oligosaccharides being identified. In Batten disease, the main subcellular location of Dol-PP-OS is within storage material, where it represents up to 7% of the dry weight. [3H]-Mannose incorporation experiments with cultured fibroblasts show that synthesis of Dol-PP-OS in JB is normal. We infer that the glycosylation intermediate Glc3Man9GlcNAc2-PP-dolichol is synthesised normally within the endoplasmic reticulum in Batten disease, but that catabolic derivatives accumulate within the lysosomes. It is unclear whether this process is central to the pathogenesis of the disease, though in IB a defect in the release of mannose residues from Dol-PP-OS is a distinct possibility.

Carbohydrate Sequence↗

PubMed1992

Derivation of the amino acid sequence of rat C-reactive protein from cDNA cloning with additional studies on the nature of its dimeric component.

Rat C-reactive protein (CRP) is unique among mammalian CRPs in being a glycoprotein and in containing a covalently linked dimer in its pentameric structure. To investigate these features, cDNA clones encoding rat CRP were isolated from an expression library, and the primary structure of the protein was derived. Taken along with the results of Northern blotting, we conclude that a single mRNA of approximately 2,500 nucleotides codes for a precursor of rat CRP with a signal sequence of 19 amino acids and a polypeptide of 211 amino acids, the latter sharing extensive homology with human, rabbit, and mouse CRPs. The deduced sequence agreed with results obtained from partial microsequencing and mapping by fast atom bombardment-mass spectrometry. Two potential sites for N-glycosylation (Asn-128 and Asn-147) and a C-terminal heptapeptide (Leu-205 to Ser-211, containing two cysteines at positions 208 and 209) were unique to rat CRP. The protein was also shown to be composed of five apparently identical monomers, two of which form a dimer linked by two interchain disulfide bonds involving Cys-208 and Cys-209. These same cysteines form an intrachain disulfide bond in the other three monomers. The primary structure of rat CRP and the basis of dimer formation have, therefore, been elucidated.

Amino Acid Sequence↗