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Carbohydrate dependent adhesion of leukocytes and the role of fucosyltransferase VII
Linköping University, Department of Biomedicine and Surgery. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Galectins, E-and P-selectins are carbohydrate-binding proteins that are up regulated at inflammatory lesions. Selectins expressed ou the activated endothelium mediate transient binding to leukocyte ligands that require sequential action of α 2,3-sialyltransferases and a 1,3-fucosyltransferases. ln leukocytes α1,3 fucosyltransferase VII adds fucose to α 2,3- sialylated lactosarnine acceptors in the final step of the biosynthesis of the selectin binding epitope sialyl Lewis x.

The finding of low sialyl Lewis x expression in polymorphonuclear leukocytes from a patient with ulcerative colitis led to the discovery of a missense mutation (G329A) in the human gene coding for α1,3 fucosyltransferase VII, FUT7. Studies including enzymatic and immunochemical analysis oftransfected cell lines and isolated polymorphonuclear leukocytes from patients confirmed that this mutation impair the ability of α1,3 fucosyltransferase VII to synthesize sialyl Lewis x. The frequency of the mutation were measured in a mixed Swedish population and found to be -1 %. One individual carrying the FUT7-G329A mutation homozygously was identified. This individual suffered from ulcer disease, noninsulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome, but the relationship between disease and the mutation is not established.

Studies using an in vitro flow chamber assay showed that transfected B-lymphoma cells and Epstein-Barr virus transformed B cells only transcribing FUT7-G329A were not able to interact with E-selectin during flow whereas polymorphonuclear leukocytes from the FUT7-G329A homozygous individual interacted well with both P- and E selectin during flow. The mRNA level of the fucosyltransferase IV coding gene, FUT4, was found to be elevated in the homozygous individual, which resulted in elevated levels ofthe CD65s epitope. However, transfected B-lymphoma cells and Epstein- Barr virus transformed cells did not show a similar elevation of FUT4 mRNA. In in vitro studies galectin-1 and- 3 were observed to be able to recruit polymorphonuclear leukocytes during flow. This thesis gives further insight into the molecular mechanisms leading to carbohydrate dependent dynamic adhesion between polymorphonuclear leukocytes and lectins during inflammation.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 41 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 762
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25551Local ID: 9999ISBN: 91-7373-204-4 (print)OAI: oai:DiVA.org:liu-25551DiVA: diva2:245881
Public defence
2003-03-28, Berzeliussalen, Universitetssjukhuset, 13:00 (Swedish)
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-24Bibliographically approved
List of papers
1. Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene
Open this publication in new window or tab >>Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene
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2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 34, 31575-31582 p.Article in journal (Refereed) Published
Abstract [en]

The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLex) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLex and SLex-related epitopes. One individual showed lower levels of SLex expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg110 → Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. TheFUT7 Arg110 is conserved in all previously cloned vertebrate α1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of ≤1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLex and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47286 (URN)10.1074/jbc.M104165200 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
2. Polymorphonuclear Leukocytes from Individuals Carrying the G329A Mutation in the α1,3-Fucosyltransferase VII Gene (FUT7) Roll on E- and P-Selectins
Open this publication in new window or tab >>Polymorphonuclear Leukocytes from Individuals Carrying the G329A Mutation in the α1,3-Fucosyltransferase VII Gene (FUT7) Roll on E- and P-Selectins
2002 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 169, no 7, 3940-3946 p.Article in journal (Refereed) Published
Abstract [en]

We recently identified several individuals carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding fucosyltransferase VII. This enzyme is involved in the biosynthesis of the sialyl Lewis x (Lex) epitope on human leukocytes, which has been identified as an important component of leukocyte ligands for E- and P-selectin. No enzyme activity was measurable in expression studies in COS-7 cells using the mutated FUT7 construct. One of the identified individuals carried this mutation homozygously. Flow cytometry analysis of polymorphonuclear leukocytes (PMN) from this individual showed a nearly complete absence of staining with mAbs directed against sialyl Lex and a diminished staining with an E-selectin IgG chimera. However, staining with P-selectin IgG chimera and Abs directed against P-selectin glycoprotein ligand-1 was not affected by the mutation. PMN from the homozygously mutated individual was further analyzed in an in vitro flow chamber assay. The number of rolling PMN and the rolling velocities on both E- and P-selectin were in the range of PMN from nonmutated individuals. FUT4 and FUT7 mRNA was quantified in PMN isolated from individuals carrying the FUT7 mutation. It was found that PMN from both FUT7 homozygously and heterozygously mutated individuals exhibited an elevated expression of FUT4 mRNA compared with PMN from FUT7 nonmutated individuals. The elevated expression of fucosyltransferase IV was reflected as an increased expression of the Lex and CD65s Ags on PMN from these individuals. The significance of the mutation was supported by transfection of BJAB cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24924 (URN)9329 (Local ID)9329 (Archive number)9329 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
3. EBV-transformed B-cells from an individual homozygously mutated (G329A) in FUT7 do not roll on E-selectin
Open this publication in new window or tab >>EBV-transformed B-cells from an individual homozygously mutated (G329A) in FUT7 do not roll on E-selectin
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The α1,3 fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x on human leukocytes. Recently, individuals were characterized carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding this enzyme. The mutated FUT7 construct was not able to produce an active Fuc-TVII enzyme in transfection studies and polymorphonuclear granulocytes from an individual carrying the mutation homozygously showed severely reduced expression of sialyl Lewis x. In the present study we have established Epstein-Barr virus transformed B-celllines from this individual (SIGN) and from an individual not carrying the mutation (IWO). The cell lines were confirmed to be of B-cell origin by flow cytometry analysis. IWO cells interacted with E-selectin in an in vitro flow chamber analysis whereas SIGN cell did not. However, when SIGN cell were transfected with wild type FUT7 cDNA interaction with E-selectin could be restored. Cell surface expression of the sLex related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared to SIGN cells, consistent with a role of these antigens in E-selectin recognition. Despite high expression of PSGL-1 neither of the cell lines interacted with P-selectin under flow. These cell lines may be useful in the further characterization ofEselectin ligands and the obtained results encourage further studies on the consequences of the FUT7-G329A mutation in vivo.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81861 (URN)
Funder
Swedish Research Council, 0002Swedish Research Council, 8266
Available from: 2012-09-24 Created: 2012-09-24 Last updated: 2012-09-24Bibliographically approved
4. Galectin mediated tethering and arrest of neutrophils under shear flow conditions
Open this publication in new window or tab >>Galectin mediated tethering and arrest of neutrophils under shear flow conditions
(English)Manuscript (preprint) (Other academic)
Abstract [en]

According to the conventional wisdom leukocyte recruitment to an inflammatory site is initiated by selectin mediated capture and rolling along the activated endothelium. The galectins are members of another family oflectins with affinity for ß-galactosides. Expressed on endothelial cells, galectin-1 and galectin-3 have been proposed to mediate cell-cell interactions during inflammation and tumor cell metastasis, and hence act as an alternative to selectins/integrins under certain circumstances. To begin testing this hypothesis, we examined the interaction of neutrophils -with a galectin-1 or galectin-3 coated surface under shear flow conditions. Both galectins were found to trigger neutrophil arrest in a dose and carbohydrate dependent manner at coating concentrations of ≥ 200 nM, and 1 dynes/cm2 wall shear stress. While, galectin-3 mediated neutrophil arrest was immediate, galectin-1 triggered a brief period of tethering before arrest. Rapidly following arrest neutrophils spread onto the galectin-coated surface. Cell spreading was accompanied by a redistribution of actin filaments, from an initial even staining with FITC-phalloidin to a more peripheral distribution in spread cells. These data suggest that galectin-1 and galectin-3 may act as adhesion molecules capturing and arresting neutrophils at sites knovvn to be less dependent on selectins and ß2integrins. They behave in part like selectins in capturing the neutrophils, but also like the integrins in triggering firm adhesion and cell spreading.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81863 (URN)
Available from: 2012-09-24 Created: 2012-09-24 Last updated: 2012-09-24Bibliographically approved

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