Evidence for an immune function of lepidopteran silk proteins.

Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.

A Drosophila salivary gland mucin is also expressed in immune tissues: evidence for a function in coagulation and the entrapment of bacteria.

Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp150, an ecdysone-regulated mucin that is found in hemocytes, the gut (peritrophic membrane) and in the salivary glands. We are particularly interested in mucin immune functions and found that gp150 is released from larval hemocytes, becomes part of the clot and participates in the entrapment of bacteria. By RT-PCR and RNAi experiments, we identified gp150 as the previously described I71-7, an ecdysone-induced salivary glue protein. We discuss the evolutionary and biochemical implications of the dual use of salivary proteins for immune functions in insects. Further molecular characterization of such shared proteins may enable a better understanding of the properties of proteins involved in containment and elimination of microbes, as well as hemostasis and wound repair.

Proteomic analysis of the Drosophila larval hemolymph clot.

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.