| 1. |
- Berglund, Jennie, et al.
(författare)
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Wood hemicelluloses exert distinct biomechanical contributions to cellulose fibrillar networks
- 2020
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Gaunitz, Stefan, et al.
(författare)
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Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins
- 2014
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 31:2, s. 145-159
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Sia alpha 2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG(2b) fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 beta 1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG(2b) is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG(2b) carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG(2b) mucin-type fusion protein carrying Sia alpha 2-3Gal as a multivalent inhibitor of influenza virus is discussed.
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| 3. |
- Gaunitz, Stefan
(författare)
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Studies on O-glycosylation of mucin-type proteins and their binding to antibodies, bacterial toxins and viral receptors
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Carbohydrates are ubiquitous on the surface of all cells in mammals where they are involved in interactions with the surroundings (extracellular matrix), other cells (including self and non-self) and microbes (bacteria and virus). Carbohydrate-protein interactions in nature are often mediated via multivalent binding where the combined strength of multiple receptor- ligand interactions results in a binding that is highly specific and strong. In this thesis we have produced proteins that are densely decorated with carbohydrate determinants in order to study the glycosylation capacity of cell lines (paper I) and generate efficient binders of antibodies (paper II), bacterial toxins (paper III) and virus receptors such as the influenza hemagglutinin (paper IV). P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-type protein that is heavily substituted with O-glycans. PSGL-1 genetically fused to mouse IgG2b Fc forms a dimeric PSGL-1/mIgG2b mucin-type fusion protein. In paper I, PSGL-1/mIgG2b was produced in Sf9 (Spodoptera frugiperda) and Hi-5 (Trichoplusia ni) cell lines. The mucin-type protein was used as a probe to analyze the O-glycosylation capacity of these cell lines, which today are used for the commercial production of recombinant proteins and vaccine components. Liquid chromatography-mass spectrometry (LC-MS) revealed that the O-glycosylation was more abundant and complex than previously reported which may limit their use for the production of therapeutic proteins. The glycosylation of PSGL-1/mIgG2b may be tailored by producing the protein in genetically engineered cell lines. Rational glycan design is achieved by transfecting cells with plasmids encoding PSGL-1/mIgG2b together with specific glycosyltransferases that expand the glycosylation capacity of the cells. In paper II, genetically engineered Chinese Hamster Ovary (CHO) cells were used to produce PSGL-1/mIgG2b carrying blood group A and B determinants on type 1, 2 and 3 outer core saccharide chains. The multivalent mucins could adsorb chain type-specific anti-A antibodies, which indicate a prospective use of the mucins in immunoadsorption (IA) columns. IA columns are used to remove anti-A and anti-B reactive antibodies prior to organ transplantation across the blood group ABO barrier. In paper III and IV, genetically engineered CHO cells were used to produce high affinity binders of Shiga toxin 1 and 2 (Stx1 and Stx2) and avian influenza hemagglutinin (H5). Biacore biosensor assays indicated that PSGL-1/mIgG2b carrying the blood group P1 determinant in multiple copies bound with high affinity to Stx1 and Stx2, while PSGL-1/mIgG2b presenting multiple copies of Siaα2,3Gal on different O-linked cores bound with high affinity to avian influenza H5. It remains to be shown if PSGL-1/mIgG2b can competitively inhibit and sterically block toxin and viral attachment to the cell surface. In conclusion, PSGL-1/mIgG2b carrying specific carbohydrates is a versatile tool that can be used in a range of applications where the multivalency confers a biologically relevant binding.
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| 4. |
- Gaunitz, Stefan, et al.
(författare)
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The N-glycan profile in cortex and hippocampus is altered in Alzheimer disease
- 2021
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 159:2, s. 292-304
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Tidskriftsartikel (refereegranskat)abstract
- 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. (Figure presented.).
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| 5. |
- Gizaw, Solomon T., et al.
(författare)
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Highly Sensitive O-Glycan Profiling for Human Serum Proteins Reveals Gender-Dependent Changes in Colorectal Cancer Patients
- 2019
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Ingår i: Analytical Chemistry. - : AMER CHEMICAL SOC. - 0003-2700 .- 1520-6882. ; 91:9, s. 6180-6189
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Rising, Anna, et al.
(författare)
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Systemic AA amyloidosis in the red fox (Vulpes vulpes)
- 2017
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Ingår i: Protein Science. - : WILEY. - 0961-8368 .- 1469-896X. ; 26:11, s. 2312-2318
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
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