Abstract
Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins—two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin—as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.
Highlights
In mammals, TG fulfills a variety of essential functions by catalyzing isopeptide bond formation between Lys and Gln residues to form e-(c-glutamyl) lysine bonds between appropriate substrates in a Ca2+-dependent manner [1,2,3]
RNA interference (RNAi) of the TG gene product using the ubiquitously expressed Da-GAL4 driver caused a pupal semi-lethal phenotype and abnormal morphology at 25uC, (Figures 3 and 4), indicating that TG is important for Drosophila development and morphogenesis
Wang et al reported that a TG-RNAi strain with reduced expression of TG using an ACt5C-GAL4 driver showed no morphological defects at 22uC [16]
Summary
TG fulfills a variety of essential functions by catalyzing isopeptide bond formation between Lys and Gln residues to form e-(c-glutamyl) lysine bonds between appropriate substrates in a Ca2+-dependent manner [1,2,3]. Plasma TG (factor XIII) stabilizes noncovalently associated fibrin polymers through covalent crosslinking of substituent fibrin monomers [4], and TG-1 (keratinocyte TG) crosslinks several proteins to form a thick layer of insoluble proteins, resulting in the formation of a cornified cell envelope [2]. In invertebrates, such as the crayfish Pacifastacus leniusculus and Drosophila, hemolymph coagulation depends on TG-mediated crosslinking of specific clotting proteins [5,6,7,8,9]. Wang et al provided proof for an immune function for Drosophila TG: Drosophila larvae with reduced TG levels exhibit increased mortality after septic injury and are more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria [16]
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