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Query: UNIPROT:P06889 (Mol)
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The Y. enterocolitica O:8(YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), D-mannose (Man), L-fucose (Fuc), and 6-deoxy-D-gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We previously characterized the 3'-end of the O-antigen gene cluster and identified four genes: two for GDP-Man biosynthesis, one for UDP-Gal biosynthesis, and one for O-antigen polymerase. Based on sequence similarity, Tn5-insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O-antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O-antigen biosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was isolated. 8081-R2 was complemented in trans with a cloned O-antigen gene cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significantly higher (approx. 100-fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O-antigen is a virulence factor.
Mol Microbiol 1997 Jan
PMID:Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8. 900 21

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. [9] whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al. [34]. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.
Plant Mol Biol 1997 Feb
PMID:Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica). 904 72

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.
Mol Immunol 1996 Oct
PMID:Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production. 907 Jun 63

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.
J Mol Biol 1997 Mar 28
PMID:Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design. 909 33

Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be identical with the low molecular weight antigen which elicits an early immunoglobulin M immune response in humans. A detailed study of the structures of these glycolipid antigens was performed. Radiolabelled glycolipids were extensively analysed by chemical and exoglycosidase treatments in combination with high pH anion-exchange chromatography, gel-filtration and lectin affinity chromatography. In addition, carbohydrate fragments prepared and purified from bulk preparations of unlabelled glycolipids by high performance liquid chromatography were subjected to two-dimensional 1H nuclear magnetic resonance spectroscopy, fast-atom bombardment-mass spectrometry, and methylation linkage analysis in order to elucidate the structure of T. gondii GPIs. The following structures were identified: (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha-inositol-PO4-lipid and the novel structure (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(Glc alpha 1-4GalNAc beta 1-4)Man alpha 1-4 GlcN alpha-inositol-PO4-lipid both with and without terminal ethanolamine phosphate. Evidence is provided, that only T. gondii GPIs bearing the unique glucose-N-acetylgalactosamine side branch are immunogenic in humans and that this structure is widely distributed among T. gondii isolates. Monoclonal antibodies have been characterized to recognize structures with different degrees of side-chain modification. We suggest that these reagents in combination with recently devised techniques for insertional mutagenesis in T. gondii should greatly facilitate the cloning of genes essential for GPI side-chain modification.
J Mol Biol 1997 Mar 07
PMID:Molecular structure of the "low molecular weight antigen" of Toxoplasma gondii: a glucose alpha 1-4 N-acetylgalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic. 910 70

The amino-terminal oxygen-binding unit Rta of the Rapana thomasiana hemocyanin is a glycoprotein with a carbohydrate content of 4.8% (w/w). Sugar analysis revealed as monosaccharide constituents xylose, fucose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. On subtracting the carbohydrate contribution from the molecular mass of 49,698 Da, determined by laser desorption mass spectrometry for Rta, an M(r) value of 47,318 Da was determined for the polypeptide part of the functional unit. The Rapana hemocyanin oxygen-binding unit Rta contains 400 residues in a single polypeptide chain. The nearly complete amino acid sequence (about 90%) is determined. This is the first report on a sequence of a marine gastropod oxygen-binding unit and also on a molluscan hemocyanin amino-terminal unit. Comparison of the Rta sequence with those of other molluscan hemocyanin units, localized in the C-terminus or in the middle of the respective multidomain polypeptide chains, revealed 42-46% homology (52-55%, including isofunctional residues). Probably, all molluscan oxygen-binding units evolved from a common ancestral gene.
Comp Biochem Physiol B Biochem Mol Biol 1997 May
PMID:Amino-terminal oxygen-binding functional unit of the Rapana thomasiana grosse (gastropod) hemocyanin: carbohydrate content, monosaccharide composition and amino acid sequence studies. 918 18

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.
Plant Mol Biol 1997 May
PMID:The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin. 920 45

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase. The resulting lysosomal storage of heparan sulfate may lead to severe neurodegeneration preceded by progressive dementia, often combined with aggressive and hyperactive behaviour. A total of 109 patients from four different geographic areas were screened for the common mutation R245H and two other previously identified mutations. SSCP analysis of exons was used to characterize the unknown alleles. We identified 16 novel sequence variants, 12 of them likely to be pathogenic. The majority of the pathogenic variants were single base pair changes leading to missense mutations. Several single base pair deletions/insertions and one nonsense mutation were also identified. Altogether, we were able to characterize 55% of the pathogenic alleles. Sequence homology between sulfamidase and N-acetylgalactosamine 4-sulfatase, the first sulfatase to have its tertiary structure defined, suggests that amino acid residues R74 and T79, which were found to be mutated, are likely to be involved in the formation of the active site of sulfamidase. R245H accounts for 31% of the Sanfilippo A alleles in Australasia, for 19.2% of the alleles in patients from the UK and has a high frequency of 57.8% in patients from The Netherlands. The identification of mutations common in certain geographic regions or ethnic groups will help in the diagnosis of MPS IIIA and allow carrier testing and improved genetic counselling.
Hum Mol Genet 1997 Sep
PMID:Novel mutations in Sanfilippo A syndrome: implications for enzyme function. 928 96

A mucin-type glycoprotein (GP) from cultured embryonic cells of Drosophila melanogaster was isolated and used to raise monoclonal antibodies (MAbs). Epitope(s) recognized by MAbs were sensitive to the treatment by O-glycanase, which specifically cleaves off O-linked mucin-type Gal(beta 1,3)GalNAc disaccharide, representing the major part of the carbohydrate moiety of Drosophila GP. Using high-affinity MAbs against carbohydrate epitopes of the Drosophila mucin GP we demonstrated its accumulation in culture medium, as well as in cultured cells, which proved to be regulated by 20-hydroxyecdysone. Mucin GPs carrying Gal(beta 1,3)GalNAc disaccharide recognized by the MAbs were immunochemically localized in several Drosophila tissues of ectodermal, mesodermal and germ line origin, including epidermal and follicle cells capable of their secretion.
Insect Biochem Mol Biol 1997 Jun
PMID:Insect mucin-type glycoprotein: immunodetection of the O-glycosylated epitope in Drosophila melanogaster cells and tissues. 930 93

Metabolic labelling of Plasmodium falciparum parasites with [3H]GlcN, [3H]Man, [3H]Gal and [3H]ethanolamine, and subsequent purification by SDS-PAGE of the labelled material provided effective labelling of the MSP-1, 195 kDa, and MSP-2, 42-53 kDa, glycoproteins. Reductive beta-elimination of the MSP-2 released from the gel consisted of glycopeptides containing labelled sugars. Processing of the eliminated components and identification of the sugar residues demonstrated the presence of N-acetylglucosaminitol and N-acetylgalactosaminitol amongst other labelled sugars. Reductive beta-elimination with sodium hydroxide-sodium borotritide-borohydride showed the presence of glucosaminitol and alanine in the hydrolysis products. The MSP-2 was retained on solid phase wheat-germ agglutinin and was released from the lectin by treatment with GlcNAc. Upon treatment with O-glycanase the MSP-2 glycoprotein released labelled amino sugar, and derived oligosaccharides on treatment with exoglycosidases released labelled components corresponding to the metabolically incorporated sugars. Labelled Gal was incorporated into the MSP-2 glycoprotein using [3H]UDP-Gal and galactosyltransferase. The galactosylated glycoprotein released labelled Gal upon treatment with beta-galactosidase. The results of the present study suggest that the carbohydrate chains of the MSP-2 glycoprotein are attached to the protein backbone via GlcNAc- and GalNAc-serine/threonine in O-glycosyl linkage and the glycoprotein has terminal GlcNAc and Gal residues. The carbohydrate moieties of MSP-2, glycoprotein consist mainly of short chains linked to the protein core.
Biochem Mol Biol Int 1997 Oct
PMID:Carbohydrate moiety of Plasmodium falciparum glycoproteins: the nature of the carbohydrate-peptide linkage in the MSP-2 glycoprotein. 935 84


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