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Proteomics of the Drosophila hemolymph clot and the function of transglutaminase
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Ulrich Theopold)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insects rely on a fast and effective coagulation and wound response to avoid loss of body fluids and immobilize pathogens. Arthropod coagulation is in some respect equivalent to vertebrate coagulation but most factors and the regulation of coagulation systems seem not to be phylogenetically conserved. To get a more complete picture of insect clotting we studied the molecular and functional nature of Drosophila hemolymph coagulation.

We developed new proteomic methods to collect Drosophila clotting factors. Several candidate factors were identified, including both predicted and novel clot proteins. Five putative TG (transglutaminase) substrates were found and we could also demonstrate that the clot is involved in immobilization of bacteria. Further investigating the role of TG we found TG to be important for Drosophila coagulation and that Fondue is a major substrate of the enzyme. Using fon RNAi knockdown we showed that Fondue affects the physical properties of the clot. A fon-GFP fusion construct was generated to follow its expression. The cuticle and the clot were labelled suggesting that Fondue is incorporated into both cuticle and clot. Clot properties and composition were affected by inhibiting TG chemically (MDC) and genetically (RNAi). Moreover, interaction between Fondue and Eig71Ee was demonstrated. Previous results indicated that coagulation could have an immune function. In hemolymph preparations, containing selected microorganisms, small deposits were seen on the microbial surfaces. The contents of these were investigated, revealing the presence of procoagulants. The targeting of microbes is instant and depends on TG and its substrates. Entomopathogenic nematode infections were performed to validate the functional importance of TG. TG RNAi knockdown larvae showed increased mortality, supporting an immune function for TG. Altogether, our data provide a more comprehensive picture of Drosophila immunity, and may further improve the understanding of innate immunity in general.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University , 2009. , 31 p.
Keyword [en]
Drosophila, clotting, transglutaminase, innate immunity
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
URN: urn:nbn:se:su:diva-26382ISBN: 978-91-7155-847-3OAI: diva2:209659
Public defence
2009-05-08, De Geer-salen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 13:00 (English)
Available from: 2009-04-16 Created: 2009-03-23 Last updated: 2010-01-14Bibliographically approved
List of papers
1. Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method
Open this publication in new window or tab >>Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method
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2004 (English)In: Current Biology, ISSN 0960-9822, Vol. 14, 625-629 p.Article in journal (Refereed) Published
urn:nbn:se:su:diva-14030 (URN)10.1016/j.cub.2004.03.030 (DOI)
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2010-01-14Bibliographically approved
2. Proteomic Analysis of the Drosophila Larval Hemolymph Clot
Open this publication in new window or tab >>Proteomic Analysis of the Drosophila Larval Hemolymph Clot
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2004 (English)In: The Journal of Biological Chemistry, Vol. 279, no 50, 52033-52041 p.Article in journal (Refereed) Published
urn:nbn:se:su:diva-14025 (URN)10.1074/jbc.M408220200 (DOI)
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2009-03-30Bibliographically approved
3. Fondue and transglutaminase in the Drosophila larval clot
Open this publication in new window or tab >>Fondue and transglutaminase in the Drosophila larval clot
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2008 (English)In: Journal of insect physiology, ISSN 0022-1910, E-ISSN 1879-1611, Vol. 54, no 3, 586-592 p.Article in journal (Refereed) Published
Abstract [en]

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.

Drosophila, Fondue, Transglutaminase, Coagulation, Immunity
National Category
Research subject
urn:nbn:se:uu:diva-87484 (URN)10.1016/j.jinsphys.2007.12.008 (DOI)000255292900007 ()18222466 (PubMedID)
Jämförande Fysiologi
Available from: 2008-12-19 Created: 2008-12-19 Last updated: 2011-03-04Bibliographically approved

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