| 1. |
- de Oliveira, Felipe Marques Souza, et al.
(författare)
-
Detection of post-translational modifications using solid-phase proximity ligation assay.
- 2018
-
Ingår i: New biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 45:October, s. 51-59
-
Tidskriftsartikel (refereegranskat)abstract
- Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.
|
|
| 2. |
- Acharya, Shikha, 1986, et al.
(författare)
-
Reduced sialyl-Lewis(x) on salivary MUC7 from patients with burning mouth syndrome
- 2019
-
Ingår i: Molecular Omics. - : Royal Society of Chemistry (RSC). - 2515-4184. ; 15:5, s. 331-339
-
Tidskriftsartikel (refereegranskat)abstract
- We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay. Presence of sialyl-Lewis(x) (Si-Le(x)) epitope on MUC7 was confirmed using Western blot. MUC7 O-glycans and measured inflammatory biomarkers were found to be similar between BMS and controls. However, oligosaccharides sialyl-Lewis(x) (Si-Le(x)) was found to be reduced in samples from BMS patients. Positive correlation (combined patients and controls) was found between levels of C-C motif chemokine 19 (CCL-19) and the amount of core-2 oligosaccharides on MUC7 as well as fractalkine (CX3CL1) and level of sialylation. Patients with BMS were shown to represent a heterogeneous group in terms of inflammatory biomarkers. This indicates that BMS patients could be further stratified on the basis of low-level inflammation. The results furthermore indicate that reduced sialylation of MUC7, particularly Si-Le(x), may be an important feature in patients with BMS. However, the functional aspects and potential involvement in immune regulation of Si-Le(x) remains unclear. Our data suggests a chemokine driven alteration of MUC7 glycosylation.
|
|
| 3. |
- Adamczyk, Barbara, 1985, et al.
(författare)
-
Sample handling of gastric tissue and O-glycan alterations in paired gastric cancer and non-tumorigenic tissues
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Sample collection, handling and storage are the most critical steps for ensuring the highest preservation of specimens. Pre-analytical variability can influence the results as protein signatures alter rapidly after tissue excision or during long-term storage. Hence, we evaluated current state-of-the-art biobank preservation methods from a glycomics perspective and analyzed O-glycan alterations occurring in the gastric cancer tissues. Paired tumor and adjacent normal tissue samples were obtained from six patients undergoing gastric cancer surgery. Collected samples (n = 24) were either snap-frozen or heat stabilized and then homogenized. Glycans were released from extracted glycoproteins and analyzed by LC-MS/MS. In total, the relative abundance of 83 O-glycans and 17 derived structural features were used for comparison. There was no statistically significant difference found in variables between snap frozen and heat-stabilized samples, which indicated the two preservation methods were comparable. The data also showed significant changes between normal and cancerous tissue. In addition to a shift from high sialylation in the cancer area towards blood group ABO in the normal area, we also detected that the LacdiNAc epitope (N, N'-diacetyllactosamine) was significantly decreased in cancer samples. The O-glycan alterations that are presented here may provide predictive power for the detection and prognosis of gastric cancer.
|
|
| 4. |
- Aspberg, A., et al.
(författare)
-
Cartilage oligomeric matrix protein forms protein complexes with synovial lubricin via non-covalent and covalent interactions
- 2017
-
Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 25:9, s. 1496-1504
-
Tidskriftsartikel (refereegranskat)abstract
- Objective: Understanding the cartilage surface structure, lost in arthritic disease, is essential for developing strategies to effectively restore it. Given that adherence of the lubricating protein, lubricin, to the cartilage surface is critical for boundary lubrication, an interaction with cartilage oligomeric matrix protein (COMP) was investigated. COMP, an abundant cartilage protein, is known to be important for matrix formation. Design: Synovial fluid (SF) from arthritic patients was used to detect possible COMP-lubricin complexes by immunological methods. Recombinant (RC) COMP and lubricin fragments were expressed to characterize this bonding and mass spectrometry employed to specifically identify the cysteines involved in inter-protein disulfide bonds. Results: COMP-lubricin complexes were identified in the SF of arthritic patients by Western blot, co-immunoprecipitation and sandwich ELISA. RC fragment solid-phase binding assays showed that the C-terminal (amino acids (AA) 518-757) of COMP bound non-covalently to the N-terminal of lubricin (AA 105-202). Mass spectrometry determined that although cysteines throughout COMP were involved in binding with lubricin, the cysteines in lubricin were primarily focused to an N-terminal region (AA 64-86). The close proximity of the non-covalent and disulfide binding domains on lubricin suggest a two-step mechanism to strongly bind lubricin to COMP. Conclusion: These data demonstrate that lubricin forms a complex network with COMP involving both non-covalent and covalent bonds. This complex between lubricin and the cartilage protein COMP can be identified in the SF of patients with arthritis conditions including osteoarthritis (OA) and rheumatoid arthritis (RA). (C) 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
|
|
| 5. |
- Axelsson, Magnus A. B., et al.
(författare)
-
Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.
- 2001
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:8, s. 633-44
-
Tidskriftsartikel (refereegranskat)abstract
- Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.
|
|
| 6. |
- Baeckström, Dan, 1956, et al.
(författare)
-
Purification and characterization of sialyl-Le(a)-carrying mucins of human bile; evidence for the presence of MUC1 and MUC3 apoproteins.
- 1994
-
Ingår i: The Journal of biological chemistry. - 0021-9258. ; 269:20, s. 14430-7
-
Tidskriftsartikel (refereegranskat)abstract
- Purification of sialyl-Le(a)-carrying mucins from primary human bile by trichloroacetic acid precipitation, delipidation, and gel filtration in guanidinium chloride gave three separable fractions, one of which was further purified by affinity chromatography. These fractions, named SBG1 (for soluble bile glycoprotein), SBG2, and SBG3 had molecular masses of > 1100, 800-950, and 100-250 kDa, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their mucin characteristics were indicated by a high carbohydrate content, ranging from 74 to 95%. The carbohydrate compositions indicated the presence of very long fucosylated polylactosamine chains. Amino acid analyses showed high abundance of serine and threonine in all three fractions (19-36%), confirming their mucin-like nature. Immunochemical analyses of deglycosylated samples detected the MUC1 mucin apoprotein in SBG2 and the MUC3 protein in SBG1. To our knowledge, this is the first report of a MUC3 mucin being purified. This mucin showed no significant reduction in size upon trypsin treatment or disulfide bond reduction and alkylation. Gel filtration of three samples of secondary bile showed that the size distribution of sialyl-Le(a)-carrying glycoproteins was similar to that found in primary bile, and immunochemical analysis showed that the MUC1 protein was present in all three samples. In one sample an additional fraction was isolated, which was insoluble in 6 M guanidinium chloride, but was solubilized upon reduction and alkylation. mRNAs from gallbladder epithelia were analyzed in Northern blot hybridizations showing that the MUC1 and MUC3 but not the MUC2 mucin apoprotein genes were expressed.
|
|
| 7. |
- Campbell, MP, et al.
(författare)
-
UniCarbKB: Putting the pieces together for glycomics research
- 2011
-
Ingår i: Proteomics. - : Wiley. - 1615-9853. ; 11:21, s. 4117-4121
-
Tidskriftsartikel (refereegranskat)abstract
- Despite the success of several international initiatives the glycosciences still lack a managed infrastructure that contributes to the advancement of research through the provision of comprehensive structural and experimental glycan data collections. UniCarbKB is an initiative that aims to promote the creation of an online information storage and search platform for glycomics and glycobiology research. The knowledgebase will offer a freely accessible and information-rich resource supported by querying interfaces, annotation technologies and the adoption of common standards to integrate structural, experimental and functional data. The UniCarbKB framework endeavors to support the growth of glycobioinformatics and the dissemination of knowledge through the provision of an open and unified portal to encourage the sharing of data. In order to achieve this, the framework is committed to the development of tools and procedures that support data annotation, and expanding interoperability through cross-referencing of existing databases. Database URL: http://www.unicarbkb.org.
|
|
| 8. |
- Flowers, Sarah A., et al.
(författare)
-
Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking
- 2020
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 295:47, s. 16023-16036
-
Tidskriftsartikel (refereegranskat)abstract
- The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.
|
|
| 9. |
- Issa, SMA, et al.
(författare)
-
Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis
- 2011
-
Ingår i: ELECTROPHORESIS. - : Wiley. - 0173-0835. ; 32:24, s. 3554-3563
-
Tidskriftsartikel (refereegranskat)abstract
- Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.
|
|
| 10. |
- Karlsson, Niclas G., 1966, et al.
(författare)
-
Analysis of monosaccharide composition of mucin oligosaccharide alditols by high-performance anion-exchange chromatography.
- 1995
-
Ingår i: Analytical biochemistry. - : Elsevier BV. - 0003-2697. ; 224:2, s. 538-41
-
Tidskriftsartikel (refereegranskat)abstract
- A simple one-step method for the analysis of monosaccharides including galactosaminitol after acidic hydrolysis is described. The hydrolyzate was re-N-acetylated and analyzed by high-performance anion-exchange chromatography and the sugars were detected by a pulsed amperometric detector. The method was applied on a mixture of neutral oligosaccharides released from mucin glycopeptides of rat small intestine by alkaline borohydride. Sugars were detected down to the nanomole range and the results were compared with monosaccharide compositional analysis performed by gas chromatography of acetylated alditols.
|
|
