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.

Figure 1. Stage-specific expression of TG.

(A) The wild-type flies were collected at indicated developmental stages and homogenized. TG antigens were detected by Western blotting (upper panel). β-tubulin was detected by Western blotting as a control (lower panel) with a mouse anti-tubulin antibody. (B) TG activity was assayed by the incorporation of Bi-PA into N, N'-dimethylcasein. The means ± S. D. of three independent experiments were plotted.

Figure 2. The effect of wounding on TG expression.

Wild-type flies were injured using a steel pin. Flies were collected at the indicated times and homogenized. (A) TG antigens at the indicated times were detected by Western blotting (upper panel). β-tubulin was detected by Western blotting as control (lower panel). (B) The amount of TG after the wounding was determined by enzyme-linked immunosorbent assay. The means ± S. D. of three independent experiments were plotted. A significant difference (versus 0 h) is indicated by asterisk (P<0.05 after Bonferroni correction). (C) TG activities were measured by the monodansylcadaverine incorporation at 1 and 4 h after wounding. The means ± S. D. of three independent experiments were plotted. A significant difference (versus 0 h) is indicated by asterisk (P<0.05).

Figure 3. Phenotypes of TG-RNAi flies.

(A) TG antigen from whole body extract of adult TG-RNAi flies was detected by Western blotting. w¹¹¹⁸, Da-GAL4>+ and Da-GAL4>UAS-LacZ IR were used as controls. Da, Da-GAL4. (B) Phenotypes of TG-RNAi flies for the wing (left panels) and abdominal cuticle (right panels) were classified into three grades depending on the extent of observed abnormality. The ratios of abnormal flies to total adult flies are indicated. Each fly was laid at 25°C. (C) Scanning electron microscopy of TG-RNAi fly. Scale bar = 200 µm.

Figure 4. The life span of the Da-GAL4>UAS-TG IR flies.

(A) The life span of the RNAi flies was compared with those of the control flies, Da-GAL4>+, Da-GAL4>UAS-lacZ IR and +>UAS-TG IR. Sixty adult flies were collected and maintained at 25°C. The number of surviving flies was recorded daily. The means ± S. D. of four independent experiments were plotted. Da, Da-GAL4. (B) Phenotypes of Tub-GAL80^ts; Da-GAL4>UAS-TG IR. Tub-GAL80^ts; Da-GAL4 flies were crossed with the UAS-TG IR flies in 20 vials and maintained at 18°C. The suppression of TG by RNAi was triggered by increasing the temperature to 29°C. The ratios flies with abnormal wings (square) and abnormal abdominal cuticles (circle) to total adult flies are indicated (upper panel). The number of adult flies born from each vial is indicated (lower panel).

Figure 5. Identification of TG substrates associated with cuticle formation.

The wings of wild-type and Da-GAL4>UAS-TG IR flies were collected at indicated times after eclosion. Wing proteins were extracted and subjected to SDS-PAGE. TG antigen was detected by Western blotting (upper panels). Loaded proteins were stained with Coomassie Brilliant Blue R-250 (lower panels). Da, Da-GAL4.

Figure 6. Phenotypes of TG substrate RNAi flies.

Phenotypes of the MS1096-GAL4>UAS-Cpr97Eb IR (A), MS1096-GAL4>UAS-Clect27 IR (B), Da-GAL4>UAS-LSP2 IR (D) and Da-GAL4>UAS-Cpr76Bd IR (E) flies. The control flies, MS-GAL4>+ (C) and Da-GAL4>+ (F), are also indicated. Each fly was laid at 25°C. MS, MS1096-GAL4; Da, Da-GAL4.

Figure 7. TG-dependent incorporation of Bi-PA to Cpr97Eb and Clect27 proteins.

Cpr97Eb (A) and Clect27 (B) proteins were incubated with or without Bi-PA in the presence of TG. The incorporation of Bi-PA was detected with biotinylated streptavidin-HRP (upper panel). Z2A (C) is a recombinant version of horseshoe crab β-1,3-D-glucan-binding protein , which was used as negative control for the TG-dependent incorporation. Loaded recombinant proteins were detected by Western blotting with an anti-6×His tag antibody or anti-Z2A antibody (lower panels).

Figure 8. Binding of Cpr97Eb and Clect27 proteins to chitin.

Cpr97Eb and Clect27 proteins were mixed with chitin, and unbound (UB) and bound (B) fractions were subjected to SDS-PAGE. The bound fraction was eluted by 2% SDS. Z2A was used as negative control for chitin binding. Proteins were detected by Coomassie Brilliant Blue R-250 staining (Clect27 and Z2A) or by anti-6×His tag antibody (Cpr97Eb).