Roles of complex and hybrid N-glycans and O-fucose glycans in oocyte development and function.
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Acyltransferases↗
Biomedical subjects
P Stanley
Publications and source records attributed to P Stanley.
Actinomycosis infection of the finger.
Actinomycosis is a rare cause of infection in the hand. We report a case of actinomycosis infection involving proximal phalanx, with the clinical presentation mimicking that of a bony neoplasm.
Actinomycosis↗
Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells.
Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.
Adenoviridae↗
Fringe modulation of Jagged1-induced Notch signaling requires the action of beta 4galactosyltransferase-1.
Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a beta 3N-acetylglucosaminyltransferase (beta 3GlcNAcT) that transfers GlcNAc to O-fucose in epidermal growth factor-like repeats of Notch. Here we use five different Chinese hamster ovary cell glycosylation mutants to identify a key aspect of the mechanism of fringe action. Although the beta 3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAc beta 3Fuc, is not elongated. The trisaccharide, Gal beta 4GlcNAc beta 3Fuc, is the minimal O-fucose glycan to support fringe modulation of Notch signaling. Of six beta 4galactosyltransferases (beta 4GalT) in Chinese hamster ovary cells, only beta 4GalT-1 is required to add Gal to GlcNAc beta 3Fuc, identifying beta 4GalT-1 as a new modulator of Notch signaling.
Animals↗
Mechanical strength testing of compacted powders.
Alternative test specimens for the determination of the fracture stress of brittle materials (eg. compacted powders) are described and discussed, and a statistical approach to the processing of strength test data is outlined.
Compressive Strength↗
Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase.
The O-fucose modification is found on epidermal growth factor-like repeats of a number of cell surface and secreted proteins. O-Fucose glycans play important roles in ligand-induced receptor signaling. For example, elongation of O-fucose on Notch by the beta1,3-N-acetylglucosaminyltransferase Fringe modulates the ability of Notch to respond to its ligands. The enzyme that adds O-fucose to epidermal growth factor-like repeats, GDP-fucose protein O-fucosyltransferase (O-FucT-1), was purified previously from Chinese hamster ovary (CHO) cells. Here we report the isolation of a cDNA that encodes human O-FucT-1. A probe deduced from N-terminal sequence analysis of purified CHO O-FucT-1 was used to screen a human heart cDNA library and expressed sequence tag and genomic data bases. The cDNA contains an open reading frame encoding a protein of 388 amino acids with a predicted N-terminal transmembrane sequence typical of a type II membrane orientation. Likewise, the mouse homolog obtained from an expressed sequence tag and 5'-rapid amplification of cDNA ends of a mouse liver cDNA library encodes a type II transmembrane protein of 393 amino acids with 90.4% identity to human O-FucT-1. Homologs were also found in Drosophila and Caenorhabditis elegans with 41.2 and 29.4% identity to human O-FucT-1, respectively. The human gene (POFUT1) is on chromosome 20 between PLAGL2 and KIF3B, near the centromere at 20p11. The mouse gene (Pofut1) maps near Plagl2 on a homologous region of mouse chromosome 2. POFUT1 gene transcripts were expressed in all tissues examined, consistent with the widespread localization of the modification. Expression of a soluble form of human O-FucT-1 in insect cells yielded a protein of the predicted molecular weight with O-FucT-1 kinetic and enzymatic properties similar to those of O-FucT-1 purified from CHO cells. The identification of the gene encoding protein O-fucosyltransferase I now makes possible mutational strategies to examine the functions of the unusual O-fucose post-translational modification.
Amino Acid Sequence↗
Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI.
A key enzyme in regulating the maturation of N-linked glycans is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC 2.4.1.101). Lec1 CHO cells lack GlcNAc-TI activity and synthesize only the oligomannosyl class of N-glycans. By contrast, Lec1A CHO mutants have weak GlcNAc-TI activity due to the reduced affinity of GlcNAc-TI for both the UDP-GlcNAc and Man(5)GlcNAc(2)Asn substrates. Lec1A CHO mutants synthesize hybrid and complex N-glycans, albeit in reduced amounts compared to parental CHO cells. In this paper, we identify two point mutations that gave rise to the Lec1A phenotype in three independent Lec1A CHO mutants. The G634A mutation in Lec1A.2C converts an aspartic acid to an asparagine at amino acid 212, disrupting a conserved DXD motif (E(211)DD(213) in all GlcNAc-TIs) that makes critical interactions with bound UDP-GlcNAc and Mn(2+) ion in rabbit GlcNAc-TI. The C907T mutation in Lec1A.3E and Lec1A.5J converts an arginine conserved in all GlcNAc-TIs to a tryptophan at amino acid 303, altering interactions that are important in stabilizing a critical structural element in rabbit GlcNAc-TI. Correction of each mutation by site-directed mutagenesis restored their GlcNAc-TI activity and lectin binding properties to parental levels. The effect of the two amino acid changes on GlcNAc-TI catalysis is discussed in relation to the crystal structure of rabbit GlcNAc-TI complexed with manganese and UDP-GlcNAc.
Animals↗
Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters.
Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations Delta serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.
Amino Acid Sequence↗
Role of the Lewis(x) glycan determinant in corneal epithelial cell adhesion and differentiation.
In this study we demonstrate that in corneal epithelium there is cell-cell contact-regulated expression of a 145-kDa glycoprotein (GP) bearing the glycan determinant Lewis(x) (Le(x)) (Galbeta(1,4)[Fucalpha(1,3)]GlcNAc). This glycoprotein (Le(x)-GP) was expressed in confluent/contact-inhibited cultures but not in sparse cultures of corneal epithelium. In contrast, a 135-kDa glycoprotein bearing precursor, unfucosylated, lactosamine-containing glycans (Galbeta1-4GlcNAcbeta1-R) was expressed in sparse cultures. Immunofluorescence staining and confocal microscopy of confluent cultures revealed that in corneal epithelium, Le(x) antigen is located in high density at sites of cell-cell adhesion. In in vitro cell-cell adhesion assays, anti-Le(x), but not anti-sialyl-Le(x) monoclonal antibodies, inhibited the formation of corneal epithelial cell-cell adhesion. Also, when added to confluent cultures, antibodies to Le(x) disrupted the monolayer and caused tightly packed polygonal cells to round up. Analysis of the expression of Fut genes that encode alpha-1,3-fucosyltransferases, the enzymes that generate the Le(x) determinant, revealed that confluent/contact-inhibited cultures of rabbit corneal epithelium contain markedly elevated levels of Fut4 and Fut3/5/6 gene transcripts compared with sparse cultures. These data suggest that the Fut4 and Fut3/5/6 genes are targets of cell-cell contact-regulated signals and that Fut gene products direct cell-cell contact-associated expression of Le(x) on the Le(x)-GP in corneal epithelium. Immunohistochemical analysis revealed that the expression of Le(x) antigen in the epithelium of adult and developing corneas is related to the stage of differentiation of the cells. Although early differentiated cells robustly expressed Le(x), relatively undifferentiated cells did not, and the expression level was relatively low in terminally differentiated cells. Overall, these data provide evidence that a Le(x)-bearing glycoprotein plays a role through the Le(x) determinant in corneal epithelial cell-cell adhesion, and these data suggest that Le(x)-mediated cell-cell interactions contribute to mechanisms that mediate corneal epithelial cell differentiation.
Amino Acid Sequence↗
Chinese hamster ovary (CHO) cells may express six beta 4-galactosyltransferases (beta 4GalTs). Consequences of the loss of functional beta 4GalT-1, beta 4GalT-6, or both in CHO glycosylation mutants.
Six beta4-galactosyltransferase (beta4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat(-)2 line of Chinese hamster ovary cells (Gat(-)2 CHO). Two independent mutants termed Pro(-)5Lec20 and Gat(-)2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro(-)5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in beta4GalT-1 transcripts in Pro(-)5Lec20 cells. The Gat(-)2Lec20 mutant expressed beta4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the beta4GalT-1 gene coding region. Northern analysis with probes from the remaining five beta4GalT genes revealed that Gat(-)2 CHO and Gat(-)2Lec20 cells express all six beta4GalT genes. Unexpectedly, the beta4GalT-6 gene is not expressed in either Pro(-)5 or Pro(-)5Lec20 cells. Thus, in addition to a deficiency in beta4GalT-1, Pro(-)5Lec20 cells lack beta4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the beta4GalT-1(-) (Gat(-)2Lec20) and beta4GalT-1(-)/beta4GalT-6(-) (Pro(-)5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, beta4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. beta4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different beta4GalTs. They may be used in glycosylation engineering and in investigating roles for beta4GalT-1 and beta4GalT-6 in generating specific glycan ligands.
Amino Acid Sequence↗
Illness perceptions and mood in chronic fatigue syndrome.
BACKGROUND: Individual beliefs and cognitions may affect adjustment to chronic fatigue syndrome (CFS) and illness perceptions, in particular, have been reported to correlate with both disability and psychological adjustment to CFS in self-diagnosed cases. OBJECTIVES: The aim of the present study was to examine these relationships in a clinic sample of CFS patients assessed by both a physician and psychiatrist. METHOD: A sample of 173 patients referred to a multidisciplinary CFS clinic and fulfilling current operational criteria for CFS [Ann Intern Med 121 (1994) 953; J R Soc Med 84 (1991) 118.] were randomly selected from the clinic database and surveyed with the Hospital Anxiety and Depression scale, Fatigue Questionnaire and Illness Perceptions Questionnaire [J Psychosom Res 37 (1993) 147; Psychol Health 11 (1996) 431; Acta Psychiatr Scand 67 (1983) 361.]. RESULTS: A total of 126 patients responded (73% response rate). The illness perception components studied were consequences (of illness), illness identity, causes (of illness), the ability to control/cure the illness and (expected) timeline of the illness. These components accounted for 15%, 28% and 30% of the variance in levels of fatigue, depression and anxiety, respectively. Two of the illness perception components (consequences and illness identity) were stronger predictors of fatigue score than mood scores. CONCLUSIONS: These findings confirmed in a clinical sample that illness perceptions are associated with variation in both disability and psychological adjustment in CFS. Illness perceptions may have an important and long-lasting effect on adaptation to CFS, and it is necessary to have a greater understanding of their role in order to tailor effective interventions for the condition.
Adult↗
Novel mimics of sialyl Lewis X: design, synthesis and biological activity of a series of 2- and 3-malonate substituted galactoconjugates.
A series of potent inhibitors of P-selectin as potential anti-inflammatory agents is reported. These compounds are derivatives of galactocerebrosides bearing a malonate side chain in positions 2 and 3 of the galactose moiety. Based on the binding mode of sialyl Lewis X, the two acidic groups of the malonate are designed to form ionic interactions with two important lysines in the active site of P-selectin, Lys113 and Lys111. On the other hand, the 4- and 6-hydroxy groups on the galactose ring are arranged to chelate the calcium ion in the P-selectin active site. The synthesis and the biological activity of this series of compounds are described. Lead compounds having a greater potency than sialyl Lewis X are identified.
Animals↗
Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.
Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes. However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.
2-Naphthylamine↗
Mural endocarditis caused by Salmonella virchow: survival with conservative treatment.
We describe a case of endocarditis caused by Salmonella enterica serotype virchow, which was treated conservatively with antibiotics alone. It is the only reported case of survival from salmonella endocarditis with conservative treatment, and the first reported case of endocarditis caused by Salmonella virchow. The changing prevalence, virulence patterns and importance of salmonella species in endocarditis are discussed.
Journal Article↗
A novel missense mutation in lysosomal sulfamidase is the basis of MPS III A in a spontaneous mouse mutant.
Sanfilippo syndrome type III A (Mucopolysaccharidosis (MPS) III A) is a rare, autosomal recessive, lysosomal storage disease, characterized by the accumulation of heparan sulfate and the loss of function of lysosomal heparan N-sulfatase activity. The disease leads to devastating mental and physical consequences and a mouse model that can be used to explore gene therapy and enzyme or cell replacement therapies is needed. We have previously identified a mouse with low sulfamidase activity and symptoms and pathologies typical of MPS III A (Bhaumik, M., Muller, V. J., Rozaklis, T., Johnson, L., Dobrenis, K., Bhattacharyya, R., Wurzelmann, S., Finamore, P., Hopwood, J. J., Walkley, S. U., and Stanley, P. [1999] A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology 9, 1389--1396). We now show that the sulfamidase gene of the MPS III A mouse carries a novel mutation (G91A) that gives an amino acid change (D31N) likely to interfere with the coordination of a divalent metal ion in the active site of this sulfatase. This spontaneous mouse mutant is an excellent model for MPS III A in humans as this disease often arises due to a missense mutation in lysosomal sulfamidase.
Amino Acid Sequence↗
The regulation of integrin function by Ca(2+).
Integrins are metalloproteins whose receptor function is dependent on the interplay between Mg(2+) and Ca(2+). Although the specificity of the putative divalent cation binding sites has been poorly understood, some issues are becoming clearer and this review will focus on the more recent information. The MIDAS motif is a unique Mg(2+)/Mn(2+) binding site located in the integrin alpha subunit I domain. Divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I domain containing integrins. The I-like domain of the integrin beta subunit also has a MIDAS-like motif but much less is known about its cation binding preferences. The N-terminal region of the integrin alpha subunit has been modelled as a beta-propeller, containing three or four 'EF hand' type divalent cation binding motifs for which the function is ill defined. It seems certain that most integrins have a high affinity Ca(2+) site which is critical for alphabeta heterodimer formation, but the location of this site is unknown. Finally intracellular Ca(2+) fluxes activate the Ca(2+) requiring enzyme, calpain, which regulates cluster formation of leucocyte integrins.
Animals↗
A novel casein kinase 2 alpha-subunit regulates membrane protein traffic in the human hepatoma cell line HuH-7.
A previously isolated endocytic trafficking mutant (TRF1) isolated from HuH-7 cells is defective in the distribution of subpopulations of cell-surface receptors for asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin, and mannose-terminating glycoproteins. The pleiotropic phenotype of TRF1 also includes an increased sensitivity to Pseudomonas toxin and deficient assembly and function of gap junctions. HuH-7xTRF1 hybrids exhibited a normal subcellular distribution of ASGR, consistent with the TRF1 mutation being recessive. A cDNA expression library derived from HuH-7 mRNA was transfected into TRF1 cells, which were subsequently selected for resistance to Pseudomonas toxin. Sequence analysis of a recovered cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2alpha". Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2alpha expression. Consistent with this finding, the hybrids HuH-7xHuH-7 and HuH-7xTRF1 expressed equivalent amounts of total CK2alpha. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2alpha and CK2alpha' and the novel CK2alpha" isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2alpha and CK2alpha', the mutant did not express CK2alpha". Based on the genomic DNA sequence, RNA transcripts encoding CK2alpha" apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2alpha or the newly cloned CK2alpha" restored the parental HuH-7 phenotype, including Pseudomonas toxin resistance, cell-surface ASGR binding activity, phosphorylation, and the assembly of gap junctions. This study suggests that HuH-7 cells express at least three CK2alpha isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.
Amino Acid Sequence↗
The second domain of intercellular adhesion molecule-1 (ICAM-1) maintains the structural integrity of the leucocyte function-associated antigen-1 (LFA-1) ligand-binding site in the first domain.
The first domain of intercellular adhesion molecule-1 (ICAM-1) binds to the leucocyte function-associated antigen-1 (LFA-1) I domain, which contains the principal ligand-binding site of this leucocyte integrin. Whether the function of the second domain is also to directly bind LFA-1 has been unclear. Our data show that mutation in the hydrophilic EF loop of ICAM-1 domain 2 resulted in impaired binding of the isolated I domain when compared with wild-type ICAM-1. LFA-1 on T-cells also binds with reduced affinity to this ICAM-1 mutant. A hybrid construct containing the first domain of vascular cell-adhesion molecule-1 joined to domains 2-5 of ICAM-1 was unable to bind to the I domain, showing that there is no direct interaction between the second domain of ICAM-1 and the I domain. This construct was also not bound by LFA-1 expressed in T-cells. Function-blocking monoclonal antibodies that map to domain 2 of ICAM-1, implicating this domain in ligand binding, were found to act indirectly. In summary our data suggest that the second domain of ICAM-1 has a role in maintaining the structure of the LFA-1 ligand-binding site in the first domain of ICAM-1 but does not appear to have a direct role in ligand binding.
Antibodies, Monoclonal↗