Proteomic Analysis of the Drosophila Larval Hemolymph Clot

Abstract

Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.

A Cysteine-rich Protein from an Arthropod Stabilizes Clotting Mesh and Immobilizes Bacteria at Injury Sites

“…This precipitation leads to a soft clot, which is hardened through the activity of cross-linking enzymes. Recently, a proteomic study of Drosophila larval hemolymph before and after clotting has led to the isolation of several candidates for clotting factors, which include proteins with similarity to prophenoloxidase-activating proteases, two phenoloxidases, and substrates for the TGase (6). On the other hand, hemolectin, one of the most abundant hemolymph proteins in Drosophila, has been isolated and characterized as a coagulation-related factor (7).…”

Overexpression of Transglutaminase in theDrosophilaWing Imaginal Disc Induced an Extra Wing Crossvein Phenotype

“…To date, the differences in catalytic activity and substrate specificity between dTG-A and -B have not been studied. A proteomic analysis of the Drosophila larval hemolymph clot showed the presence of TG substrate proteins and TG activity in the larval hemolymph, 6) and TG was found to act in hemolymph coagulation in Drosophila.…”

The serpin Spn5 is essential for wing expansion in Drosophila melanogaster

“…A transcriptional profiling of distinct Drosophila blood cell (hemocytes) populations showed that, of the 13000 genes represented on the microarray, over 2500 exhibited significantly enriched transcription according to hemocytes subgroups; among these were genes encoding serine proteases and serpins such as Spn5 (Irving et al, 2005). Karlsson et al (Karlsson et al, 2004) and Irving et al (Irving et al, 2005) have detected of Spn5 in larval hemolymph, while De Gregorio et al, and Ligoxygakis et al, reported in flies deficient for another Serpin called Spn27A constitutive melanization of the cuticle and wings of adult flies, or melanotic tumors in larvae Ligoxygakis et al, 2002). In preliminary results, we failed to detect Spn5 mRNA in larval hemolymph cells, or aberrant activation of melanization in flies with a low basal level of Spn5 expression (data not shown).…”