Identification of collagen IV derived danger/alarm signals in insect immunity by nanoLC-FTICR MS

Abstract

The immune system can be stimulated by microbial molecules as well as by endogenously derived danger/alarm signals of host origin. Using the lepidopteran model insect Galleria mellonella, we recently discovered that fragments of collagen IV, resulting from hydrolysis by microbial metalloproteinases, represent danger/alarm signals in insects. Here, we characterized immune-stimulatory peptides generated by thermolysin-mediated degradation of collagen IV using nanospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) after separation by nanoscale liquid chromatography (nanoLC). The combination of FTICR MS analysis and de novo peptide sequencing resulted in the identification of 38 specific collagen IV fragments of which several peptides included the integrin-binding motif RGD/E known from numerous mammalian immune-related proteins. Custom-synthesized peptides corresponding either to the presently identified collagen peptide GIRGEHyp or to a well-known integrin-binding RGD peptide (GRGDS) were injected into G. mellonella to determine their immune-stimulatory activities in vivo. Both peptides stimulated immune cells and systemically the expression of lysozyme and a specific inhibitor of microbial metalloproteinases. Further examination using specific MAP kinase inhibitors indicated that MEK/ERK and p38 are involved in RGD/E-mediated immune-signaling pathways, whereas JNK seems to play only a minor role.

Lepidopteran transcriptome analysis following infection by phylogenetically unrelated polydnaviruses highlights differential and common responses

“…In Bombyx mori, immunolocalization experiments show that granulocytes discharge collagen, which attaches to foreign entities and adjoining hemocytes to facilitate binding in capsules (Adachi et al, 2005). Fragments of collagen IV generated by the activity of microbial metalloproteinases may also play a role in activating hemocytes (Altincicek et al, 2009). In S. frugiperda, 4 different transcripts encode predicted collagen IV a chains with HdIV and MdBV reducing the abundance of each (Table 3).…”

Coevolution between pathogen-derived proteinases and proteinase inhibitors of host insects

“…Similar to microbial thermolysin, the endogenous G. mellonella MMP has been shown to be capable to degrade collagen type IV resulting in formation of peptidic fragments which, in turn, induce expression of the IMPI and other antimicrobial peptides. 69 Sensing and feedback-loop regulation of microbial metalloproteinases has become a constituent of insect innate immunity. However, because the insect immune system can obviously not discriminate whether microbial or endogenous metalloproteinases degrade host proteins such as collagen IV, it makes sense to produce simultaneously two inhibitors with distinct activities.…”

“…Particular peptidic fragments resulting from hydrolysis of collagen IV turned out to represent danger signals which, in turn, can set the immune system into alarm. 66,69 Thermolysin is also a potent activator of the enzyme cascade that controls phenoloxidase activity which catalyzes the formation of melanin. Hemolymph coagulation resulting in…”

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

“…The infectious non-self model postulates that the immune system is set into alarm by recognition of microbial pattern molecules which are absent in the host, e.g., microbial cell wall components, whereas the so-called danger model explains the activation of immune response by alarm signals from injured cells, such as those exposed to pathogens, toxins or mechanical damage (Altincicek et al, 2009;Matzinger, 2002).…”