Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (>0.5 M NaCl required for elution) contained an internal IdoUA(2-OSO 3 )-GlcNSO 3 (6-OSO 3 )-IdoUA(2-OSO 3 )-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1.
Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are major components of the cell surface of Gram-negative bacteria with diverse roles in bacterial pathogenesis of animals and plants that include elicitation of host defenses. Little is known about the mechanisms of perception of these molecules by plants and about the associated signal transduction pathways that trigger plant immunity. Here we address the issue of the molecular basis of elicitation of plant defenses through the structural determination of the LOS of the plant pathogen Xanthomonas campestris pv. campestris strain 8004 and examination of the effects of LOS and fragments obtained by chemical treatments on the immune response in Arabidopsis thaliana. The structure shows a strong accumulation of negatively charged groups in the lipid A-inner core region and has a number of novel features, including a galacturonyl phosphate attached at a 3-deoxy-D-manno-oct-2-ulosonic acid residue and a unique phosphoramide group in the inner core region. Intact LOS and the lipid A and core oligosaccharides derived from it were all able to induce the defense-related genes PR1 and PR2 in Arabidopsis and to prevent the hypersensitive response caused by avirulent bacteria. Although LOS induced defense-related gene transcription in two temporal phases, the core oligosaccharide induced only the earlier phase, and lipid A induced only the later phase. These findings suggest that plant cells can recognize lipid A and core oligosaccharide structures within LOS to trigger defensive cellular responses and that this may occur via two distinct recognition events. Lipopolysaccharides (LPSs)2 are ubiquitous and vital components of the cell surface of Gram-negative bacteria (1, 2). They are amphiphilic macromolecules composed of a hydrophilic heteropolysaccharide (comprising the core oligosaccharide and O-specific polysaccharide or O-chain) covalently linked to a lipophilic moiety termed lipid A, which anchors these macromolecules to the outer membrane. LPSs not possessing the O-chain are termed rough LPSs or lipooligosaccharides (LOSs).In animal and insect cells, innate immune defenses are triggered by the perception of pathogen-associated molecular patterns (PAMPs), conserved and generally indispensable microbial structures including LPSs. The recognition of PAMPs by these cells is often mediated by leucine-rich repeat proteins such as Toll in Drosophila and the Toll-like receptors in mammals (3-6). Recognition of LPSs occurs through the lipid A moiety, which is responsible for most of the biological effects of LPS in animals. Lipid A toxicity in animals strongly depends on its structure and is also influenced by the covalently linked core region, which possesses immunogenic properties (1, 2).LPSs apparently have diverse roles in bacterial pathogenesis of plants. As major components of the outer membrane, LPSs are involved in the protection of bacterial cell, contributing to reduce the membrane permeability and thus allowing growth of bacterium in the unfavorable conditi...
The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.
The thermal decomposition pathways leading to the formation of volatile compounds and to char residue in poly(bisphenol A carbonate) (PC), poly(resorcinol carbonate) (PRC), and poly(hydroquinone carbonate) (PHC) have been investigated by mass spectrometry. The structure of the volatile compounds obtained in the temperature range 300−700 °C, by direct pyrolysis mass spectrometry (DPMS), suggests that these polycarbonates undergo thermal decomposition by a number of different pyrolysis processes. In the initial stage of the thermal degradation are generated cyclic oligomers by an intramolecular exchange reaction, whereas the evolution of CO2 and H2O is spread over all the pyrolysis temperature range, being responsible for the formation of ether bridges (decarboxylation) and phenolic end groups (hydrolysis). A disproportionation reaction of the BPA isopropylidene bridges of PC itself takes place at higher temperature yielding phenyl and isopropylidene end groups, whereas pyrolysis products containing dibenzofuran units are formed by dehydrogenation of ether units. The formation of compounds containing xanthone and fluorenone units, most likely generated by isomerization of the aromatic carbonate functional groups and successive condensation reaction, has also been observed. In an additional set of experiments, the isothermal pyrolysis of PC was achieved, isothermally, at 350 and 400 °C, and then the exhaustive and selective aminolysis of the carbonate functional groups, still present in the pyrolysis residue, has been performed. The aminolyzed residue was then analyzed by fast atom bombardment (FAB) mass spectrometry to detect the compounds eventually formed by molecular rearrangements of PC chains. The FAB-MS spectra showed the presence of compounds containing several consecutive xanthone and ether units, indicating that at this temperature the isomerization and the condensation processes leading to these structures are quite extensive. These units undergo aromatization and cross-linking processes, leading to a graphite-like charred residue as the temperature increases.
PMM2-CDG (PMM2 gene mutations) is the most common congenital disorder of N-glycosylation. We conducted a nationwide survey to characterize the frequency, clinical features, glycosylation and genetic correlates in Italian patients with PMM2-CDG. Clinical information was obtained through a questionnaire filled in by the referral physicians including demographics, neurological and systemic features, neuroimaging data and genotype. Glycosylation analyses of serum transferrin were complemented by MALDI-Mass Spectrometry (MALDI-MS). Between 1996 and 2012, data on 37 Italian patients with PMM2-CDG were collected. All the patients with a severe phenotype were unable to walk unaided, 84 % had severe intellectual disability and 81 % microcephaly. Conversely, among 17 mildly affected patients 82 % had independent ambulation, 64 % had borderline to mild intellectual disability and 35 % microcephaly. Epilepsy and stroke-like events did not occur among patients with the mild phenotype. The rate and extent of systemic involvement were more pronounced in severely affected patients. The L32R misfolding mutation of the PMM2 gene occurred in 70 % of the patients with the mild phenotype and was associated with a less severe underglycosylation of serum Tf at MALDI-MS analyses. Despite their different disease severity, all patients had progressive (olivo)ponto-cerebellar atrophy that was the hallmark clinical feature for the diagnosis. A mild neurological phenotype of PMM2-CDG marked by preserved ambulatory ability and autonomy and associated with L32R mutation is particularly frequent in Italy. PMM2-CDG should be considered in patients with even mild developmental disability and/or unexplained progressive cerebellar atrophy.
Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.
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