Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

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.

Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish

“…Drosophila TG forms clots and traps invading pathogenic microbes (10,11) and cross-links drosocrystallin on the peritrophic matrix, a semipermeable barrier structure in insects, to form a stabilized fiber structure against toxic proteases released by orally infected pathogenic bacteria (12). Moreover, RNAi directed against the TG gene in Drosophila leads to a pupal semilethal phenotype, abnormal morphology, and shorter lifespan compared with non-RNAi flies (8,13).…”

AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae

“…TGases catalyze formation of isopeptide bonds between a free ⑀-amino group and glutamine residue in a Ca 2ϩ -dependent manner (15)(16)(17) and are essential for a variety of biological processes. For instance, in the nematode Onchocerca volvulus, TGase-catalyzed cross-linking is crucial for the molting of third instar larvae (18), whereas in Drosophila, TGase1 plays an essential role in pupal morphogenesis and cuticle schlerotization brought about by cross-linking of two cuticular chitin-binding proteins, larval serum protein 2 and a putative C-type lectin (19). During immune responses in crayfish, hemolymph coagulation is achieved by TGase-mediated crosslinking of specific clotting proteins (20 -22).…”

RNA Interference Directed against the Transglutaminase Gene Triggers Dysbiosis of Gut Microbiota in Drosophila

“…UAS-TG IR flies were obtained from the National Institute of Genetics (Mishima, Japan). To obtain the TG-RNAi flies, Da-GAL4 flies were crossed with UAS-TG IR flies, and to obtain the non-TGRNAi flies, Da-GAL4 flies were crossed with w 1118 flies, as described previously (22). The fly strain Rel E20 was described previously (43).…”