Abstract
Hemicelluloses, a family of heterogeneous polysaccharides with complex molecular structures, constitute a fundamental component of lignocellulosic biomass. However, the contribution of each ...hemicellulose type to the mechanical properties of secondary plant cell walls remains elusive. Here we homogeneously incorporate different combinations of extracted and purified hemicelluloses (xylans and glucomannans) from softwood and hardwood species into self-assembled networks during cellulose biosynthesis in a bacterial model, without altering the morphology and the crystallinity of the cellulose bundles. These composite hydrogels can be therefore envisioned as models of secondary plant cell walls prior to lignification. The incorporated hemicelluloses exhibit both a rigid phase having close interactions with cellulose, together with a flexible phase contributing to the multiscale architecture of the bacterial cellulose hydrogels. The wood hemicelluloses exhibit distinct biomechanical contributions, with glucomannans increasing the elastic modulus in compression, and xylans contributing to a dramatic increase of the elongation at break under tension. These diverging effects cannot be explained solely from the nature of their direct interactions with cellulose, but can be related to the distinct molecular structure of wood xylans and mannans, the multiphase architecture of the hydrogels and the aggregative effects amongst hemicellulose-coated fibrils. Our study contributes to understanding the specific roles of wood xylans and glucomannans in the biomechanical integrity of secondary cell walls in tension and compression and has significance for the development of lignocellulosic materials with controlled assembly and tailored mechanical properties.
A newly developed microscale protocol for profiling serum O-glycans has been validated here with multiple serum samples obtained from different cohorts of colorectal cancer patients. The simultaneous ...cleavage and permethylation steps in this procedure preserve the integrity of released minor O-glycans, so that 39 O-linked oligosaccharides could be reliably recorded in a profile. This is far more detected components than shown in any previous studies. The analytical results were further subjected to a battery of statistical tests. Our O-glycan compositions compare favorably with the previous results obtained with solid tumors and cancer cell lines, suggesting that smaller circulatory mucins protruding into the blood circulation may be one source of O-glycans that we observe in the serum samples. While the control vs cancer statistical comparisons generally agree with the expected glycosylation trends, the comparisons of male vs female subjects have led to some surprising results for which we do not have a ready explanation due to lack of any literature describing hormonal control of O-glycosylation. Our results thus underscore the necessity of applying new analytical technologies to clinically interesting sample sets.
The study of exosomes has become increasingly popular due to their potentially important biological roles. Urine can be used as an effective source of exosomes for noninvasive investigations into the ...pathophysiological states of the urinary system, but first, detailed characterization of exosomal components in healthy individuals is essential. Here, we significantly extend the number of N-glycan compositions, including sulfated species, identified from urinary exosomes and determine the sialic acid linkages for many of those compositions. Capillary electrophoresis-mass spectrometry (CE-MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify N-glycan and sulfated N-glycan compositions. Second, because the alteration of sialylation patterns has been previously implicated in various disease states, ion-exchange chromatography, microfluidic capillary electrophoresis (CE), and MALDI-MS were adopted to resolve positional isomers of sialic acids. Structures of the sialyl-linkage isomers were assigned indirectly through α2–3 sialidase treatment and sialic acid linkage-specific alkylamidation (SALSA). In total, we have identified 219 N-glycan structures that include 175 compositions, 64 sialic acid linkage isomers, 26 structural isomers, and 27 sulfated glycans.
Exosomes are extracellular nanosized vesicles with lipid bilayers encapsulating nucleic acids and proteins, both with and without glycosylation. While exosomal nucleic acids and proteins have ...previously been explored to identify cancer biomarkers with some promising results, little information has been available concerning their glycoconjugate content. Exosomes were isolated from normal urine samples through multistep differential centrifugation. The isolated exosomes have an average size of 146 nm and a spherical shape, as determined by dynamic light scattering and transmission electron microscopy, respectively. N-Glycans were enzymatically released from the isolated vesicles. After being reduced and permethylated, N-glycans were measured by MALDI mass spectrometry. Paucimannosidic, high-mannose, and complex type glycans were identified and their relative abundances were determined. Some detailed structures of these glycans were revealed through liquid chromatography/tandem mass spectrometry (LC/MS-MS). The reduced N-glycans, without being permethylated, were also separated and analyzed by LC/MS-MS, and their structures were further detailed through isomeric separation on porous graphitized carbon (PGC) packed in long capillaries. Using microfractionation before LC/MS-MS, minor multiantennary N-glycans were preconcentrated as based on hydrophobicity or charge. Preconcentration of the reduced and permethylated glycans on a C18 cartridge revealed numerous large glycans, whereas fractionation of the reduced N-glycans by ion-exchange cartridges facilitated detection of sulfated glycans. After removing N-glycans from the original sample aliquot, O-glycans were chemically released from urinary exosomes and profiled, revealing some unusual structures.
Proteomics-large-scale studies of proteins-has over the last decade gained an enormous interest for studies aimed at revealing proteins and pathways involved in disease. To fully understand ...biological and pathological processes it is crucial to also include post-translational modifications in the "omics". To this end, glycomics (identification and quantification of glycans enzymatically or chemically released from proteins) and glycoproteomics (identification and quantification of peptides/proteins with the glycans still attached) is gaining interest. The study of protein glycosylation requires a workflow that involves an array of sample preparation and analysis steps that needs to be carefully considered. Herein, we briefly touch upon important steps such as sample preparation and preconcentration, glycan release, glycan derivatization and quantification and advances in mass spectrometry that today are the work-horse for glycomics and glycoproteomics studies. Several proteins related to Alzheimer disease pathogenesis have altered protein glycosylation, and recent glycomics studies have shown differences in cerebrospinal fluid as well as in brain tissue in Alzheimer disease as compared to controls. In this review, we discuss these techniques and how they have been used to shed light on Alzheimer disease and to find glycan biomarkers in cerebrospinal fluid.
Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox ...and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full‐length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter‐species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model.
To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate ...binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K m appears to be a true dissociation constant, and k cat describes the chemical reaction of the ES complex, not product release. Enzyme−substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES⧧), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k cat/K m (describing the overall conversion of the free substrate to ES⧧) is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k cat/K m) is much lower than that for the spontaneous reaction (k non) (ΔΔG ⧧ = −21.8 kcal/mol at 25 °C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S⧧), is almost entirely enthalpic in origin (ΔΔH = −20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES⧧ complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3‘-hydroxyl moiety of cytidine is disrupted by truncation of either group, k cat/K m and transition state affinity are each reduced by a factor of 104. This effect of mutation is entirely enthalpic in origin (ΔΔH ∼ 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES⧧ complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.
Protein glycosylation is crucial for the central nervous system and brain functions, including processes that are defective in Alzheimer disease (AD) such as neurogenesis, synaptic function, and ...memory formation. Still, the roles of glycans in the development of AD are relatively unexplored. Glycomics studies of cerebrospinal fluid (CSF) have previously shown altered glycosylation pattern in patients with different stages of cognitive impairment, including AD, compared to healthy controls. As a consequence, we hypothesized that the glycan profile is altered in the brain of patients with AD and analyzed the asparagine‐linked (N‐linked) glycan profile in hippocampus and cortex in AD and control brain. Glycans were enzymatically liberated from brain glycoproteins and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Eleven glycans showed significantly different levels in hippocampus compared to cortex in both control and AD brain. Two glycans in cortex and four in hippocampus showed different levels in AD compared to control brain. All glycans that differed between controls and AD brain had similar structures with one sialic acid, at least one fucose and a confirmed or potential bisecting N‐acetylglucosamine (GlcNAc). The glycans that were altered in AD brain differed from those that were altered in AD CSF. One glycan found to be present in significantly lower levels in both hippocampus and cortex in AD compared to control contained a structurally and functionally interesting epitope that we assign as a terminal galactose decorated with fucose and sialic acid. Altogether, these studies suggest that protein glycosylation is an important component in the development of AD and warrants further studies.
The N‐glycan profile of brain in Alzheimer‐ disease (AD) was studied by glycomics analysis of homogenates from hippocampus (Hip) and frontal cortex (Ctx) and compared with nondemented controls. N‐glycans were enzymatically released and separated from the glycoproteins, labeled with 2‐anthranilic acid, purified and analyzed by liquid chromatography‐tandem mass spectrometry. The levels of four N‐glycans in hippocampus and two in frontal cortex, all with confirmed structures from mass spectrometric fragmentation, were significantly altered in AD compared to control. These findings may lay ground for novel potential treatment strategies.
Alzheimer disease (AD) is a devastating disease and a global health problem, and current treatments are only symptomatic. A wealth of clinical studies support that the disease starts to develop ...decades before the first symptoms appear, emphasizing the importance of studying early changes for improving early diagnosis and guiding toward novel treatment strategies. Protein glycosylation is altered in AD but it remains to be clarified why these alterations occur and how they affect the disease development. Here, we used a glycomics approach to search for alterations in protein glycosylation in cerebrospinal fluid (CSF) in AD compared with nondemented controls. Using both matrix‐assisted laser desorption ionization‐time of flight and liquid chromatography–electrospray mass spectrometry, we observed an increase in N‐glycans carrying bisecting N‐acetylglucosamine in AD. Based on those findings, we designed an enzyme‐linked multiwell plate assay to quantify N‐glycans binding to the lectin Phaseolus vulgaris Erythroagglutinin (PHA‐E), which is specific for N‐glycans containing bisecting N‐acetylglucosamine. Using this assay, we found a similar increase in CSF in AD compared with controls. Further analysis of CSF from 242 patients with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), or AD dementia revealed significantly increased binding to PHA‐E in MCI and AD compared to SCI. Interestingly, PHA‐E binding correlated with CSF levels of phosphorylated tau and total tau and this correlation was most prominent in the SCI group (R = 0.53–0.54). This study supports a link between N‐glycosylation, neurodegeneration, and tau pathology in AD and suggests that glycan biomarkers have potential to identify SCI cases at risk of developing AD.
Glycomics analyses showed alterations in the N‐glycan pattern in cerebrospinal fluid (CSF) in Alzheimer disease as compared to controls. The levels of several N‐linked glycans carrying bisecting N‐acetylglucosamine were increased in AD. A multiwell plate enzyme‐linked lectin assay denoted enzyme‐linked lectin assay (ELLA) was developed based on these findings. Intriguingly, a correlation between ELLA data and tau levels was observed both in AD and in subjective cognitive impairment.
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