Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

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

Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species

Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion

The life span of the <i>Da-GAL4>UAS-TG IR</i> flies.

<p>(A) The life span of the RNAi flies was compared with those of the control flies, <i>Da-GAL4>+</i>, <i>Da-GAL4>UAS-lacZ IR</i> and <i>+>UAS-TG IR</i>. 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. <i>Da</i>, <i>Da-GAL4</i>. (B) Phenotypes of <i>Tub-GAL80^ts; Da-GAL4>UAS-TG IR</i>. <i>Tub-GAL80^ts; Da-GAL4</i> flies were crossed with the <i>UAS-TG IR</i> flies in 20 vials and maintained at 18°C. The suppression of <i>TG</i> 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).</p

Identification of TG substrates associated with cuticle formation.

<p>The wings of wild-type and <i>Da-GAL4>UAS-TG IR</i> 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). <i>Da, Da-GAL4.</i></p

Phenotypes of TG substrate RNAi flies.

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