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Role of N-linked glycosylation in expression of E-selectin on human endothelial cells
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Hospital Pharmacy, University Hospital, Linköping, Sweden.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
1995 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 25, no 9, 2452-2459 p.Article in journal (Refereed) Published
Abstract [en]

E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.

Place, publisher, year, edition, pages
1995. Vol. 25, no 9, 2452-2459 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84628DOI: 10.1002/eji.1830250907OAI: oai:DiVA.org:liu-84628DiVA: diva2:560845
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Innate immune response in human endothelial cells: characterization and regulation of E-selectin, ICAM-I and cytokine expression and the role of Staphylococcus aureus
Open this publication in new window or tab >>Innate immune response in human endothelial cells: characterization and regulation of E-selectin, ICAM-I and cytokine expression and the role of Staphylococcus aureus
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Endothelial cells located on the vessel walls play an important role in inflammation. In response to cytokines or other inflammatory mediators such as microorganisms, endothelial cells express cell adhesion molecules such as E-selectin and ICAM-1 that are involved in binding circulating leukocytes to the endothelium. Since E-selectin is highly glycosylated one aim of this thesis was to investigate the role of N-glycosylation in E-selectin expression and function. Modification of glycosylation was obtained by culturing HUVEC in the presence of soluble glycosylation inhibitors. The result showed that E-selectin is highly glycosylated with N-Iinked, complex type oligosaccharides which were found to be important for the level of cell surface expression and turnover time. However, defect glycosylation did not affect its binding properties as revealed in a static cell-binding assay. Combinations of cytokines can produce a different expression of E-selectin. When TNF-α and IFN-γ were used to stimulate HUVEC an enhanced and prolonged expression of E-selectin was obtained compared to when HUVEC were stimulated with TNF-α alone. This effect could be blocked by monensin, which is an inhibitor of trans-Golgi network processing. This again indicated a role for posttranslational modification in regulation of E-selectin expression.

Eighteen clinical isolates of Staphylococcus aureus were analysed for their ability to induce expression of E-selectin and ICAM-1 in HUVEC. Upregulation of adhesion molecules varied between isolates; 17 isolates induced expression of E-selectin and 13 of ICAM-1. Some isolates induced a significant expression of E-selectin without stimulation of ICAM-1, whereas the opposite was not found. Bacterial viability was required for induction of the adhesion molecules. There was no correlation between ability of S. aureus to induce expression of cell adhesion molecules and methicillin susceptibility, pulse field electrophoresis patterns, biochemical characteristics, phage typing or toxin production. Nine cytokines, IL-1ß, TNF-α, IL-6, IL-8, IL-10, IL-12p70, GRO-α, GM-CSF and RANTES, all of importance in the inflammatory process, were analysed in supernatants from HUVEC stimulated with eighteen isolates of S. aureus. All isolates induced IL-6, IL-8, GRO-α, GM-CSF and RANTES. Isolates of S. aureus inducing high expression of one of these cytokines also showed high expression of the other four, indicating a possible common mechanism for regulation of the expression of these cytokines. The ability of individual S. aureus isolates to induce expression of cytokines correlated with their ability to induce expression of ICAM-1 but not E-selectin in HUVEC. No correlation between cytokine profile and S. aureus production of enterotoxin A-D, TSST-1, cytotoxicity, PFGE- and phage pattern or susceptibility to methicillin was observed. Furthermore, culture filtrate from S. aureus induced expression of ICAM-1 and IL-8 in HUVEC. The component(s) from culture filtrates were heat-stable, had a molecular weight of about 100 kDa and the effect was independent of the presence of fetal calf serum in media.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 52 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 804
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26669 (URN)11235 (Local ID)91-7373-493-4 (ISBN)11235 (Archive number)11235 (OAI)
Public defence
2003-09-26, Elsa Brändströmsalen, Hälsouniversitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-16Bibliographically approved

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Påhlsson, PeterStrindhall, JanLundblad, Arne

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