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B W Gibson

Publications and source records attributed to B W Gibson.

At least 55 records · Page 3Linked to original sources
PubMed1994

Lipooligosaccharide biosynthesis in pathogenic Neisseria. Cloning, identification, and characterization of the phosphoglucomutase gene.

The lipooligosaccharide (LOS) of pathogenic Neisseria is an important factor in disease pathogenesis. Little is known about the genes involved in neisserial LOS biosynthesis. To elucidate specific LOS biosynthetic genes, we screened a Tn916 library that was constructed in Neisseria meningitidis strain NMB. This strain expresses a single LOS that has an molecular mass of 4.5 kDa and binds monoclonal antibody (mAb) 3F11. This library was screened using a mAb panel that recognizes structural differences in neisserial LOS oligosaccharides. A stable LOS mutant of strain NMB was identified which we designated NMB-R6. This mutant expressed an LOS with an molecular mass of approximately 3.1-3.2 kDa and did not bind mAb 3F11. Genomic DNA from this mutant transformed N. meningitidis strain NMB to the tetracycline resistant NMB-R6 phenotype greater than 10(-4)/recipient/micrograms of DNA. In addition, we transformed Neisseria gonorrhoeae strain 1291 (LOS phenotype molecular mass 4.5 kDa, mAb 3F11+) to the NMB-R6 LOS phenotype with N. meningitidis NMB-R6 genomic DNA. Analysis of N. gonorrhoeae strain 1291-R6 LOS by mass spectroscopy showed that the LOS oligosaccharide structure is GlcNAc-->Hep2phosphoethanolamine-->2-keto-3-deoxymannooctuloson ic acid (where Hep is heptose). Sequence analysis showed that the transposon is inserted into the 3' end of a gene that has homology to the human phosphoglucomutase (PGM) gene. Sequence comparison indicated that the putative PGM gene in N. gonorrhoeae 1291 and N. meningitidis NMB had 92% identity at the DNA level. PGM and glucokinase activity was present in cell free extracts of N. meningitidis NMB and N. gonorrhoeae strain 1291. N. meningitidis NMB-R6 and N. gonorrhoeae strain 1291-R6 had no detectable PGM activity, whereas glucokinase activity was similar to the wild type strains. PGM activity can be reconstituted in N. meningitidis strain NMB-R6 by transformation with the cloned PGM gene. SDS-polyacrylamide gel electrophoresis demonstrated that NMB-R6 transformed with the PGM gene expressed the 3F11+, 4.5-kDa LOS of the parent NMB strain. The inability of N. meningitidis NMB-R6 and N. gonorrhoeae strain 1291-R6 to convert glucose 6-phosphate to glucose 1-phosphate results in the truncated LOS phenotype expressed by these mutants.

Amino Acid Sequence↗
PubMed1994

Meningococcal group A lipooligosaccharides (LOS): preliminary structural studies and characterization of serotype-associated and conserved LOS epitopes.

Structural studies indicate that the neisserial lipooligosaccharides (LOS) are composed of an oligosaccharide (OS) portion with a phosphorylated diheptose (Hep) core attached to the toxic lipid A moiety. A conserved meningococcal LOS epitope, defined by monoclonal antibody (MAb) D6A, is expressed on group A and many group B and C meningococci of different LOS serotypes (J. J. Kim, R. E. Mandrell, H. Zhen, M. A. Apicella, J. T. Poolman, and J. M. Griffiss, Infect. Immun. 56:2631-2638, 1988). This MAb-defined D6A epitope is immunogenic in humans (M. M. Estabrook, R. E. Mandrell, M. A. Apicella, and J. M. Griffiss, Infect. Immun. 58:2204-2213, 1990; M. M. Estabrook, C. J. Baker, and J. M. Griffiss, J. Infect. Dis. 197:966-970, 1993). In this study, we characterize this important MAb-defined LOS epitope. Serotype L10 and L11 group A meningococal LOS were chemically modified and used to investigate what portion of the LOS molecule is important for expression of the conserved (D6A) epitope and serotype-associated LOS epitopes by use of immunoblotting techniques and selected MAbs as probes. Preliminary structural characterization of the LOS was also accomplished by electrospray ionization-mass spectrometry. Our results indicate the following. (i) Antibodies that recognize the serotype-associated or conserved LOS epitopes recognize the OS portion of the LOS. (ii) The phosphorylated diheptose core region of the OS is essential for expression of the conserved D6A epitope. (iii) The lipid portion of the molecule is important for optimum expression of the LOS epitopes. (iv) The proposed compositions of the O-deacylated LOS are consistent with the presence of a phosphorylated diheptose core and are as follows: for O-deacylated L10 LOS, 3Hex (hexose), 1HexNAc (N-acetylhexosamine), 2KDO (2-keto-3-deoxy-D-manno-octulosonic acid), 2Hep (heptose), 1PEA or 2PEA (phosphoethanolamine), and O-deacylated lipid A; and for O-deacylated L11 LOS, 2Hex, 1HexNAc, 2KDO, 2Hep, 2PEA, and O-deacylated lipid A. Because the phosphorylated diheptose core region of the LOS is essential for the formation of a conserved LOS epitope (D6A) that is immunogenic in humans, care should be taken to maintain stereochemical requirements for the expression of this conserved epitope in the design of effective, nontoxic LOS vaccines.

Animals↗
PubMed1994

Use of pyocin to select a Haemophilus ducreyi variant defective in lipooligosaccharide biosynthesis.

Haemophilus ducreyi, a cause of genital ulcer disease in developing countries, appears to facilitate the heterosexual transmission of the human immunodeficiency virus in Africa. Despite an increase in studies of this gram-negative human pathogen, little is known about the pathogenesis of chancroid. Our studies have shown that the lipooligosaccharides (LOS) of H. ducreyi may play an important role in ulcer formation. Monoclonal antibody and mass spectrometric analyses identified a terminal trisaccharide present on H. ducreyi LOS that is immunochemically similar to human paragloboside. This epitope is present on the LOS of Neisseria gonorrhoeae, and it may be the site of attachment for pyocin lysis. We have used pyocin, produced by Pseudomonas aeruginosa, to select LOS variants with sequential saccharide deletions from N. gonorrhoeae. On the basis of the similarities between N. gonorrhoeae and H. ducreyi LOS, we employed the same technique to determine if H. ducreyi strains were susceptible to pyocin lysis. In this study, we report the generation of a pyocin N-resistant H. ducreyi strain which synthesizes a truncated version of the parental LOS. Further studies have shown that this H. ducreyi variant has lost the terminal LOS epitope defined by monoclonal antibody 3F11. This report demonstrates that H. ducreyi is sensitive to pyocins and that this technique can be used to generate H. ducreyi LOS variants. Such variants could be used in comparative studies to relate LOS structure to biologic function in the pathogenesis of chancroid.

Carbohydrate Sequence↗
PubMed1993

Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing.

The only component of the infectious scrapie prion identified to date is a protein designated PrPSc. A posttranslational process converts the cellular PrP isoform (PrPC) into PrPSc. Denatured PrPSc was digested with endoproteases, and the resulting fragments were isolated by HPLC. By both mass spectrometry and Edman sequencing, the primary structure of PrPSc was found to be the same as that deduced from the PrP gene sequence, arguing that neither RNA editing nor protein splicing feature in the synthesis of PrPSc. Mass spectrometry also was used to search for posttranslational chemical modifications other than the glycosylinositol phospholipid anchor attached to the C-terminus and two Asn-linked oligosaccharides already known to occur on both PrPSc and PrPC. These results contend that PrPSc molecules do not differ from PrPC at the level of an amino acid substitution or a posttranslational chemical modification; however, we cannot eliminate the possibility that a small fraction of PrPSc is modified by an as yet unidentified posttranslational process or that PrPC carries a modification that is removed in the formation of PrPSc. It seems likely that PrPSc differs from PrPC in its secondary and tertiary structure, but the possibility of a tightly bound, disease-specific molecule which purifies with PrPSc must also be considered.

Amino Acid Sequence↗
PubMed1993

Structural studies of the lipooligosaccharides from Haemophilus influenzae type b strain A2.

The outer membrane lipooligosaccharides (LOS) from Haemophilus influenzae type b strain A2 are a heterogeneous mixture of glycolipids containing a conserved Lipid A structure and a variable oligosaccharide moiety. After O-deacylation by treatment with anhydrous hydrazine, the O-deacylated LOS mixture was analyzed by electrospray mass spectrometry and shown to contain 11 components, ranging in M(r) from 2277.8 to 3416.4. The majority of these structures contained a variable number of hexoses, three L-glycero-D-manno-heptoses, and one 3-deoxy-D-manno-octulosonic acid (KDO) residue attached to a diphosphorylated O-deacylated Lipid A moiety. Additional phosphate and phosphoethanolamine (PEA) groups were also present on the oligosaccharide structures. Two minor high molecular weight components were also observed that contained N-acetylhexosamine and sialic acid. Neuraminidase treatment of the O-deacylated LOS mixture resulted in the loss of sialic acid from these latter two species. After mild acid hydrolysis and separation by size-exclusion chromatography, liquid secondary ion mass spectrometry identified six major and four minor oligosaccharides, ranging in M(r) from 1243.4 to 2215.8. These released oligosaccharides contained a common heptose trisaccharide core structure with anhydro-KDO at the reducing terminus, which arises as an artifact of the hydrolysis procedure by beta-elimination of a phosphate group from the 4-position of KDO. Selected oligosaccharide fractions were subjected to composition and methylation analyses and sequenced by tandem mass spectrometry. Taken together, these data defined the major O-deacylated LOS as follows: [formula: see text] Higher molecular weight structures in the mixture contained galactose, N-acetylglucosamine, and sialic acid as additional branch sugars, suggesting that H. influenzae A2 is capable of forming a sialylated lactosamine structure.

Carbohydrate Sequence↗
PubMed1993

Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry.

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.

Antigens, Bacterial↗
PubMed1992

Partial characterization of the major lipooligosaccharide from a strain of Haemophilus ducreyi, the causative agent of chancroid, a genital ulcer disease.

The first preliminary structure of a surface lipooligosaccharide from Haemophilus ducreyi has been determined. The major oligosaccharide was released by mild acid hydrolysis and analyzed by liquid secondary ion and tandem mass spectrometry. The mass spectral data combined with composition and methylation analysis yielded the most probable structure; Gal1----4GlcNAc1----3Gal1----4Hep1----6Glc1----( Hep1----2Hep1----)3,4Hep1---- KDO, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or KDO) exists in an anhydro form. This anhydro species results from the elimination of a phosphate from C-4 of KDO during mild acid hydrolysis. The core heptose trisaccharide consists of L-glycero-D-manno-heptose, but analysis of the peracetylated sugars indicated that the 1,4-linked heptose is likely D-glycero-D-manno-heptose. The monoclonal antibody 3F11 generated against Neisseria gonorrhoeae also binds to this lipooligosaccharide and suggests that the terminal trisaccharide is Gal beta 1----4GlcNAc beta 1----3Gal beta 1----, an epitope found in the glycose moiety of the human erythrocyte glycosphingolipid lactoneotetraglycosylceramide. Mass spectrometric and composition analysis of the lipid A moiety shows that it is similar to the lipid A of Haemophilus influenzae strain I-69 Rd-/b+ proposed by Helander et al. (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). Electrospray mass spectrometric analysis of the intact O-deacylated lipooligosaccharides gave an average Mr of 2710, and supported an overall structure consisting of the above nonasaccharide linked directly to a diphosphorylated lipid A moiety through the single KDO which is phosphorylated. This structure should provide a framework to investigate the roles of lipooligosaccharides in the host immunochemical response and pathology of H. ducreyi infection, a leading cause of genital ulcer disease.

Carbohydrate Sequence↗
PubMed1992

Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae.

Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.

Antigens, Bacterial↗
PubMed1992

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.

Amino Acid Sequence↗
PubMed1992

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs. The presence in shark liver of an FABP which differs substantially in primary structure from mammalian liver FABP, while being closely related to the FABP expressed in mammalian heart muscle, peripheral nerve myelin and adipocytes, opens a further dimension regarding the question of the existence of structure-dependent and tissue-specific specialization of FABP function in lipid metabolism.

Amino Acid Sequence↗
PubMed1992

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.

Carbohydrate Sequence↗
PubMed1991

Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis.

The structures and hemolytic and bactericidal activities of three bombinin-like peptides, or BLP-1-3, from the skin of Bombina orientalis are described. The peptides were isolated from the skin of B. orientalis and sequenced by tandem mass spectrometry and are amphipathic, cationic peptides of 25-27 amino acids in length. The sequence of the most abundant member (BLP-1) is: Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu- Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NH2. All three peptides were found to share considerable, but not complete, homology with bombinin, an antimicrobial, hemolytic peptide first isolated by Michl and Csordas (Csordas, A., and Michl, A. (1970) Monatsh. Chem. 101, 182-189) from the skin of Bombina variegata. The BLPs have been assayed for antibiotic and hemolytic activity and found to be more potent than magainin 2 (a related antimicrobial peptide from Xenopus laevis) in their ability to kill bacteria. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic over eukaryotic membranes. The molecular basis for antibacterial activity is presumed to be due to their predicted amphipathic alpha-helical structures which is supported by circular dichroism measurements that found significant helical content (63-69% alpha-helix) in 40% trifluoroethanol. Last, a cDNA library was constructed from the skin of B. orientalis and screened with an oligonucleotide probe complementary to the COOH terminus of BLP-1. Several clones were isolated and sequenced that encode BLP-1 and BLP-3, as well as an additional peptide (BLP-4) that differs by two amino acid substitutions from BLP-3.

Amino Acid Sequence↗
PubMed1991

The structural basis for pyocin resistance in Neisseria gonorrhoeae lipooligosaccharides.

Pyocin resistance in a strain of Neisseria gonorrhoeae has been found to be associated with structural differences in the oligosaccharide moieties of the gonococcal outer membrane lipooligosaccharides (LOS). N. gonorrhoeae strain 1291 had been treated with several pyocins, usually lethal bacteriocins produced by Pseudomonas aeruginosa, and a series of surviving mutants were selected. The LOS of these pyocin-resistant mutants had altered electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gels (Dudas, K. C., and Apicella, M. A. (1988) Infect. Immun. 56, 499-504). Structural analyses of the oligosaccharide portions of the wild-type (1291 wt) and five pyocin-resistant strains (1291a-e) by liquid secondary ion mass spectrometry, tandem mass spectrometry, and methylation analysis revealed that four of the mutant strains make oligosaccharides that differ from the wild-type LOS by successive saccharide deletions (1291a,c-e) and, in the oligosaccharide of 1291b, by the addition of a terminal Gal to the 1291c structure. The composition, sequence, and linkages of the terminal tetrasaccharide of the wild-type LOS are the same as the lacto-N-neotetraose terminus of the human paragloboside (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-ceramide), and both glycolipids bound the same monoclonal antibodies O6B4/3F11 that recognize this terminal epitope. None of the pyocin-resistant mutants bound this antibody. The 1291b LOS bound a monoclonal antibody that is specific for Gal alpha 1----4Gal beta 1----4Glc-ceramide (Pk glycosphingolipid) and shared a common composition, sequence, and linkages with this latter glycosphingolipid. Organisms that bound the anti-Pk monoclone occurred at the rate of approximately 1/750 among the wild-type parent strain. This structural information supports the conclusion that treatment with pyocin selects for mutants with truncated LOS structures and suggests that the oligosaccharides contained in the LOS of the wild-type strain and 1291b mimic those of human glycosphingolipids.

Carbohydrate Sequence↗
PubMed1991

Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis.

Monoclonal antibodies (MAb) 3F11 and 06B4 recognize epitopes that are conserved on gonococcal lipooligosaccharides (LOS), present on some meningococcal LOS, and conserved on human erythrocytes. LOS of some group B and C prototype meningococcal LOS strains (LOS serotypes L1 to L8) treated with neuraminidase showed increased expression of the 3F11 and 06B4 MAb-defined epitopes. Neuraminidase-treated LOS separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stained showed a shift in migration from a component with a mass of approximately 4.8 kDa to a component with a mass of between 4.5 and 4.6 kDa. The same strains grown in medium with excess CMP-N-acetylneuraminic acid had LOS that shifted in migration to a slightly higher component (mass, approximately 4.8 kDa). Chemical analysis of the neuraminidase-digested products from one LOS indicated it contained approximately 1.5% sialic acid. Covalent linkage between sialic acid and the LOS was confirmed by analysis of de-O-acylated and dephosphorylated LOS by liquid secondary ion mass spectrometry. Three studies show that some meningococci contain sialic acid in their LOS, that the sialic acid is cleaved and lost in conventional acetic acid hydrolysis, and that the sialic acid alters the expression of MAb-defined epitopes.

Antibodies, Monoclonal↗
PubMed1990

Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein.

Permethylation of the glycan isolated from the glycoinositol phospholipid (GPI) anchor of the scrapie prion protein (PrPSc) trimethylates a free hexosamine to form a quarternary ammonium salt, substantially increasing the sensitivity for analysis by mass spectrometry. This derivatization induces specific fragmentation reactions in collision-induced dissociation spectra obtained on a four-sector tandem mass spectrometer, identifying the branching pattern of the PrPSc GPI glycan.

Glycolipids↗
PubMed1990

Structural models for the cell surface lipooligosaccharides of Neisseria gonorrhoeae and Haemophilus influenzae.

A structural model is proposed for the surface glycolipids, or lipooligosaccharides (LOS), of gram-negative pathogenic bacteria that colonize human mucosae, e.g. Neisseria gonorrhoeae and Haemophilus influenzae. The development of this model has involved analysis of a series of pyocin-resistant mutants with altered LOS and other recent immunochemical and structural data. A comprehensive approach to determining the necessary structural data has been constructed that utilizes liquid secondary ion mass spectrometry, tandem mass spectrometry, methylation analysis and nuclear magnetic resonance. To prepare purified oligosaccharides for these analyses, chromatographic and chemical techniques have been developed that include high-pH anion-exchange chromatography of underivatized oligosaccharides and reverse-phase chromatography after derivatization with hydrazino alkyl benzoates. The proposed LOS model has several unique features that distinguish it from models developed for the lipopolysaccharides of enteric bacteria. This information should lead to an understanding of the unique structure/function relationship of LOS and to the development of carbohydrate-based vaccines.

Chromatography, Gel↗
PubMed1990

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5.

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b5 were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from a Staphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariush et al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b5 were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2 +/- 2 and 9502.3 +/- 2 for bovine and rabbit trypsin-solubilized cytochrome b5, respectively. In both cases, these molecular weights correspond to a cytochrome b5 fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b5 was found to be 10,144.5 +/- 2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1-88) of Mr 10,146.1.

Amino Acid Sequence↗