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Effects of α1‐acid glycoprotein onpolymorphonuclear leukocytes ‐involvement of cell surface receptors
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-20271ISBN: 9789173935746 (print)OAI: oai:DiVA.org:liu-20271DiVA: diva2:233567
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
List of papers
1. 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)
Open this publication in new window or tab >>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)
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.

Keyword
Orosomucoid, plasma protein, calcium signaling, carbohydrate, L-selectin, phagocyte
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20262 (URN)10.1096/fj.07-8534com (DOI)17675532 (PubMedID)
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2009-09-09Bibliographically approved
2. Effects of alpha 1-acid Glycoprotein Fucosylation on its Ca2+ Mobilizing Capacity in Neutrophils
Open this publication in new window or tab >>Effects of alpha 1-acid Glycoprotein Fucosylation on its Ca2+ Mobilizing Capacity in Neutrophils
2009 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 69, no 5, 412-420 p.Article in journal (Refereed) Published
Abstract [en]

We recently showed that the acute-phase protein alpha(1)-acid glycoprotein (AGP) induces rises in cytosolic calcium concentration, [Ca2+](i,) in neutrophils through sialic acid dependent interactions with the neutrophil receptors siglec-5 and/or siglec-14. Whereas both siglec-5 and siglec-14 have a relatively broad specificity for sialylated oligosaccharide structures, including both structures with terminal alpha 2-3 or alpha 2-6 linked sialic acid, there is a markedly reduced affinity to the fucosylated epitope sialyl Lewis x (SLe(x)). Increased fucosylation, leading to increased expression of SLe(x) on AGP is commonly associated with inflammatory conditions. In the present study, we investigated whether an increased SLe(x) expression would affect the Ca2+-mobilizing effect of AGP. AGP with elevated fucose content isolated from patients with untreated chronic joint inflammation showed a decreased [Ca2+](i) modulatory effect on neutrophils compared to normally fucosylated AGP. Furthermore a hyperfucosylated AGP form produced by in vitro fucosylation, that consequently had an elevated expression of SLe(x), could not elicit a [Ca2+](i) increase in neutrophils. The role of the carbohydrate portion of AGP in modulating neutrophil responses was further strengthened by showing that synthetic glycoconjugates carrying oligosaccharides with terminal alpha 2-3 or alpha 2-6 linked sialic acid were able to mimic the Ca2+-mobilizing effect of AGP whereas a synthetic glycoconjugate carrying SLe(x) was not. Based on these data, we conclude that increased fucosylation can alter the ability of AGP to induce neutrophil signalling and further supports an important role of the oligosaccharide chains of AGP in the modulation of leukocyte functions during an inflammatory process.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18019 (URN)10.1111/j.1365-3083.2009.02240.x (DOI)
Note

On the day of the defence day the title of this article was "Increased fucosylation of α1‐acid glycoprotein decreases its Ca2+ mobilizing capacity in neutrophils".

Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2017-12-13Bibliographically approved
3. Alpha-1-acid glycoprotein interacts with the neutrophilproteins S100A8 and S100A9
Open this publication in new window or tab >>Alpha-1-acid glycoprotein interacts with the neutrophilproteins S100A8 and S100A9
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The acute phase protein α1-acid glycoprotein (AGP) has been indicated tobind to neutrophils and to affect neutrophil functions. In the presentinvestigation neutrophil proteins interacting with AGP was studied. Twolow molecular weight proteins were isolated from neutrophil lysates byaffinity chromatography on a column with immobilized AGP. The proteinswere identified as the calcium-binding, myeloid related proteins S100A8and S100A9 using mass spectrometry and Western blot analyses. Theinteraction was further confirmed using a biotin affinity-tagging procedure.The interaction between AGP and S100A8/A9 was sensitive to EDTAindicating a calcium-dependent binding. Desialylation andhyperfucosylation of AGP did not affect its binding to S100A8/A9.

Keyword
Neutrophils, plasma proteins, inflammation, affinity chromatography
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20266 (URN)
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2010-01-14Bibliographically approved
4. Alpha1-acid glycoprotein is a carrier of hydroxyeicosatetraenoic acids – role in calcium mobilization ofpolymorphonuclear granulocytes
Open this publication in new window or tab >>Alpha1-acid glycoprotein is a carrier of hydroxyeicosatetraenoic acids – role in calcium mobilization ofpolymorphonuclear granulocytes
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have previously shown that α1-acid glycoprotein (AGP) induces rises incytosolic calcium concentration, [Ca2+]i, in human polymorphonuclear granulocytes(PMN) and that this effect is enhanced by prior pre-sensitization of PMN with theanti L-selectin antibody DREG-56. AGP is a known carrier of several lipophilicsubstances. This study was designed to determine whether lipids bound to AGP areinvolved in the induction of [Ca2+]i mobilization in PMN. We found that delipidatedAGP elicited a smaller rise in [Ca2+]i in DREG-56 pretreated PMN compared tonative AGP and that lipids extracted from AGP provoked an increase in [Ca2+]i thatwas potentiated by L-selectin pre-engagement with DREG-56. Similarly to whatwas previously found for AGP, the increase in [Ca2+]i produced by the AGP lipidextract involved activation of src-tyrosine kinases and PI3-kinases. The AGP lipidextract was analyzed by high-performance thin layer chromatography. Individualbands were extracted from the plate and their Ca2+ mobilizing activity wasanalyzed. One band contained activity and was further analyzed by electro-spraytandem mass spectrometry. The active band contained a mixture of hydroxyeicosatetraenoic acids (HETEs) with 12-HETE being one of the major components.Pharmacological studies indicated that the AGP lipid extract acted through theleukotriene B4-receptor type II, BLT2. This study supports the hypothesis that someof the immunomodulatory properties that have been attributed to AGP may beconnected to lipids carried by this plasma protein.

Keyword
alpha1-acid glycoprotein, hydroxy eicosatetraenoic acids, BLT2, cytosolic calcium, polymorphonuclear leukocyte
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20267 (URN)
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2010-01-14Bibliographically approved

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