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The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs)
Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
2007 (English)In: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, ISSN 1530-6860, Vol. 21, no 14, 4059-4069 p.Article in journal (Refereed) Published
Abstract [en]

We studied whether the acute-phase protein alpha1-acid glycoprotein (AGP) induces rises in [Ca2+]i in neutrophils and sought to identify the corresponding AGP receptor (or receptors). We found that AGP elicited a minimal rise in [Ca2+]i in Fura-2-loaded neutrophils, and this response was markedly enhanced by pretreatment with anti-L-selectin antibodies. (The EC50 value of the AGP-induced Ca2+ response was 9 microg/ml.) Activation of phospholipase-C, Src tyrosine kinases, and PI3 kinases proved to be essential for the AGP-mediated increase in [Ca2+]i, whereas the p38 MAPK and SYK signaling pathways were not involved. Furthermore, antibodies against sialic acid binding, immunoglobulin-like lectin 5 (Siglec-5) and oligosaccharide 3'-sialyl-lactose both antagonized the AGP-induced response and caused an immediate increase in [Ca2+]i in anti-L-selectin-treated neutrophils, which indicates a signaling capacity of Siglec-5. We used modified forms of AGP (treated with mild periodate or neuraminidase) to establish the importance of sialic acid residues. The modified forms of AGP caused a much smaller rise in [Ca2+]i than did unaltered AGP. Affinity chromatography confirmed that unchanged AGP, but not neuraminidase-treated AGP, bound to Siglec-5. Our report provides the first evidence for a signaling capacity by AGP through a defined receptor. Pre-engagement of L-selectin significantly enhanced this signaling capacity.

Place, publisher, year, edition, pages
2007. Vol. 21, no 14, 4059-4069 p.
Keyword [en]
Orosomucoid, plasma protein, calcium signaling, carbohydrate, L-selectin, phagocyte
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-20262DOI: 10.1096/fj.07-8534comPubMedID: 17675532OAI: oai:DiVA.org:liu-20262DiVA: diva2:233532
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2009-09-09Bibliographically approved
In thesis
1. Effects of α1‐acid glycoprotein onpolymorphonuclear leukocytes ‐involvement of cell surface receptors
Open this publication in new window or tab >>Effects of α1‐acid glycoprotein onpolymorphonuclear leukocytes ‐involvement of cell surface receptors
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alpha1‐acid glycoprotein (AGP) is a highly glycosylated lipid‐binding acute‐phaseprotein. Although the exact mechanisms are unknown, several studies havesuggested that AGP may regulate the function of neutrophils and hence modulateinflammatory responses. The general aim of this thesis was to investigate if AGP isable to mediate intracellular signalling in neutrophils through binding to specificreceptors.

Measurements of intracellular calcium concentration showed that AGP elicited asmall rise in [Ca2+]i in neutrophils that was markedly enhanced by pre‐treatmentwith anti‐L‐selectin antibodies. In contrast, desialylation of AGP reduced the Ca2+mobilizing capacity significantly. The AGP‐induced Ca2+ signal was mediatedthrough Src tyrosine kinases, PLC and PI3K which suggests involvement of cellsurface receptors. Indeed, AGP was shown to bind to, and mediate Ca2+ signallingthrough, sialic acid binding immunoglobulin‐like lectin (Siglec)‐5 and/or ‐14.Increased fucosylation of AGP is common during acute‐phase reactions. We showthat hyperfucosylated AGP has a diminished Ca2+ signalling capacity compared tonormally fucosylated AGP. This could be due to a reduced capacity of AGP tointeract with Siglec‐5/‐14 since it is known that the presence of fucose residues onsialylated glycans has a negative effect on Siglec‐5/‐14 affinity. AGP was alsodemonstrated to bind to the neutrophil proteins S100A8 and S100A9. In additionwe show that AGP‐bound hydroxyeicasotetraenoic acids (HETEs) induce increasesin [Ca2+]i in neutrophils through binding to the leukotriene B4 receptor BLT2. Wepropose a two‐step binding model where AGP binds to Siglec‐5/‐14 on L‐selectinactivated neutrophils. This may orient AGP in a way that assists an interactionbetween AGP and the neutrophil membrane which favours transfer of AGP‐boundHETEs to the BLT2 receptor.

In conclusion, these data gives new insights regarding how AGP interacts with andmediates signalling in human neutrophils and supports the view of AGP as beingan acute phase reactant with immunomodulatory properties.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 61 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1139
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20271 (URN)9789173935746 (ISBN)
Public defence
2009-08-28, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2017-12-14Bibliographically approved
2. α1-acid glycoprotein modulates the function of human neutrophils and platelets
Open this publication in new window or tab >>α1-acid glycoprotein modulates the function of human neutrophils and platelets
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The acute-phase protein α1-acid glycoprotein (AGP; orosomucoid) was initially identified andcharacterised in the 1950s. The normal plasma concentration is around 0.5-1 mg/ml butduring inflammation the concentration increase several fold and the carbohydrate compositionof the protein changes. AGP is a highly glycosylated protein with 45 % of the molecularweight consisting of glycans. These glycans are believed to be of importance for the functionof the protein. However, the precise physiological role of AGP is still unclear.

The present thesis reveals that AGP at physiological concentration induce calcium elevationin human neutrophils and platelets. In neutrophils this response was enhanced several fold ifsurface L-selectin was pre-engaged. Our results showed that this L-selectin-mediatedamplification was abolished if the neutrophils were pre-treated with Src or phosphoinositide3-kinase (PI3K) inhibitors. AGP alone did not induce production of reactive oxygen species(ROS) in neutrophils. However, if the neutrophils were activated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) a subsequent addition of AGP caused aprominent ROS response. Moreover, both the calcium rise and the ROS response weredepending on sialic acid residues on AGP. In the case of calcium elevation we defined thereceptor as sialic-acid-binding immunoglobulin-like lectin (Siglec)-5 on the neutrophil.

In platelets, AGP induced a Rho-kinase dependent phosphorylation of myosin phosphatasetarget subunit-1 (MYPT1) and a minor calcium response. This resulted in a prominent plateletshape change (i.e. spherical shape and granule centralization) recorded as change in lighttransmission and by differential interference contrast (DIC) microscopy. The shape changecaused by AGP was strongly suppressed by inhibition of Rho-kinase and abolished by Rhokinaseinhibition combined with chelation of intracellular calcium. No other manifestations ofplatelet activation like aggregation or secretion were registered. Opposite to neutrophils theeffect of AGP on platelets was not mediated by an interaction between sialic acid and siglecmolecules. However, the results indicated that AGP may bind to a collagen/thrombospondin-1surface receptor. Endogenous inhibitors like nitric oxide (NO) and adenosine abolished theAGP-induced platelet shape change. The antagonizing action of NO on shape change causedby AGP was long acting. In comparison, other aspects of agonist-induced platelet activation(e.g. intracellular calcium elevations) are only transiently suppressed by NO. This indicatesthat endothelium-derived NO may play a crucial role to counter balance the effect of AGP in vivo.

Take together the results in this thesis reveal that AGP can initiate intracellular signalling andmodulate functional responses in neutrophils and platelets.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 65 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1135
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20476 (URN)978-91-7393-614-9 (ISBN)
Public defence
2009-09-04, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2012-02-03Bibliographically approved

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Gunnarsson, PeterLevander, LouisePåhlsson, PeterGrenegård, Magnus

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