Proteomic identification of differentially expressed and phosphorylated proteins in epidermis involved in larval-pupal metamorphosis of Helicoverpa armigera

Abstract

Background: Metamorphosis is an important process in the life cycle of holometabolous insects and is regulated by insect hormones. During metamorphosis, the epidermis goes through a significant transformation at the biochemical and molecular levels.

Results: To identify proteins and phosphoproteins involved in this process, we separated and compared epidermal protein profiles between feeding larvae and metamorphically committed larvae using two-dimensional gel electrophoresis and Pro-Q Diamond Phosphoprotein Staining. Sixty-one spots showing differential expression and/or phosphorylation were analyzed by mass spectrometry and eighteen proteins were proved related to larval-pupal transformation. Eight of them were further examined at the mRNA level by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and two of them were examined at the protein level by Western blot. Calponin was highly expressed in the metamorphic epidermis and phosphorylated by protein kinase C.

Conclusion: Our results suggest that the expression and phosphorylation of these proteins may play important roles in coordinating the biochemical processes involved in larval-pupal metamorphosis.

Figure 1

Separation of proteins from H. armigera larval epidermis by 2-DE (2nd dimension: 12% SDS-PAGE). (A) 5th feeding (5th-F) larvae with Coomassie Brilliant Blue (CBB) stain. (B) 6th-metamorphically committed (6th-M) larvae with CBB staining. (C) 5th-F larvae with Pro-Q Diamond phosphoprotein gel (Pro-Q) stain. (D) 6th-M larvae with Pro-Q stain. Spots analyzed by mass spectrometry are designated by numbers. Spots annotated with the same number in both gels are the same spots visualized by different staining methods. The experiments were replicated three times.

Figure 2

Exhibition of spots from the 5th feeding larvae (5th-F) and 6th-metamorphically committed larvae (6th-M) stained with Coomassie Brilliant Blue (CBB stain) and Pro-Q diamond phosphoprotein stain (Pro-Q stain). (A) The protein expression was up-regulated but no phosphorylation was detected; (B) The protein expression was up-regulated and phosphorylation was also detected; (C) The protein expression remained the same but the phosphorylation decreased; (D) The protein expression remained the same but the phosphorylation increased.

Figure 3

Reverse Transcription PCR analysis of transcription of genes encoding proteins identified in 2D-electrophoresis. Total RNA was isolated from 5th-feeding (5-24 h), 6th-feeding (6-24 h, 6-48 h), and 6th-metamorphically committed (6-72 h, 6-96 h, 6-108 h) larval epidermis. The β-actin gene was used for normalization of the compared templates. The gene expression ratio was calculated by Quantity One (Version 4.5, Bio-Rad, United States). All of the experiments were repeated at least three times. The values are mean ± standard deviation obtained by normalization of target genes against β-actin (*, difference is significant compared to 5-24 h by student t test, p < 0.05).

Figure 4

Western Blot analysis of protein expressions in epidermis. (A) Expression of hexamerin (82 kD) and calponin (19 kD) in the epidermis during larval development. 5th-F, fifth feeding larvae; 6th-F, sixth feeding larvae; 6th-M, 6th instar metamorphically committed larvae. 5-24 h, 6-24 h, 6-48 h, 6-72 h, 6-96 h and 6-120 h respectively denote samples from different developmental stages. (B) Effect of 20E injection on the expression of hexamerin and calponin in the epidermis. Same diluted DMSO was used as a control. 6-6 h, 6-12 h, 6-24 h and 6-48 h are the developmental ages of larvae. (C) Analysis of phosphorylation of calponin, same diluted DMSO as a control; 20E, sample from 6-12 h larvae injected with 20E; λPPase, sample from same 20E-injected larvae and treated with λPPase; CC+20E, sample from 6-12 h larvae injected with CC and 20E.

Figure 5

Developmental profiles and hormone induction of calponin and Br in epidermis. (A) Expression changes of both genes. 5-24 h, 5-36 h, 6-0 h, 6-24 h, 6-48 h, 6-72 h, 6-96 h, 6-120 h, P-0 day denote mRNA samples from different developmental stages from 5th-instar 24 hour to pupae. (B) 20E induction. 6th-6 h larvae were injected with 20E (500 ng/larva) for 1 h, 3 h, 6 h, 12 h and 24 h, DMSO injection as a control.