The Insect Hemolymph Protein HP19 Mediates the Nongenomic Effect of Ecdysteroids on Acid Phosphatase Activity

Abul Arif,Palaniappan Vasanthi,Immo Alex Hansen,Klaus Scheller,Aparna Dutta-Gupta

doi:10.1074/jbc.m402311200

Abstract

The activity of acid phosphatase (ACP) in insect fat bodies is stimulated by the steroid hormone 20-hydoxyecdysone (20E) in vivo. However, in fat bodies kept in culture, a factor from the hemolymph is required to enhance the ACP activity. We identified the factor as a protein with a molecular mass of 19 kDa (HP19) from the hemolymph of a lepidopteran insect, the rice moth, Corcyra cephalonica. Western analysis of hemolymph proteins with denaturing and non-denaturing PAGE using antibodies raised against HP19 suggest that this protein exists as a monomer. It is synthesized by the hind gut-associated lobular fat body of the larvae and is released into the hemolymph. The stimulatory effect of HP19 on the ACP activity is developmentally regulated and exhibits its maximal effect shortly before the onset of metamorphosis. We cloned the HP19 cDNA by immunoscreening a hind gut-associated lobular fat body cDNA expression library. Analysis of the amino acid sequence shows that HP19 belongs to the family of glutathione S-transferase (GST) like proteins. However, affinity-purified GST from Corcyra failed to show any mediation effect on 20E-stimulated ACP activity, and HP19 lacks GST enzymatic activity. Notably, HP19 mediates the hormone-stimulated ACP activity in intact fat body tissue and homogenates even in the presence of inhibitors of transcription and translation, suggesting a nongenomic mode of action. In addition, we show that HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor protein.

Highlights

Insect metamorphosis, the transition from the larval to the adult stage of insects, is controlled by ecdysteroid hormones [1,2,3]
Identification of a Protein in the Hemolymph of Corcyra That Mediates the 20E-stimulated acid phosphatase (ACP) Activity in the Fat Bodies of Thorax-ligated, Hormone-deprived Larvae—When insect larvae are ligated behind the first pair of prolegs, i.e. behind the hormone producing glands, the posterior part of the animal is known to be relatively free of endogenous ecdysteroids [35,36,37]
Because the ACP activity in the fat body was significantly lower after 24 h of ligation, this time period was used for all

Summary

EXPERIMENTAL PROCEDURES

Insects—Larvae of the rice moth, C. cephalonica (Stainton), were reared on coarsely crushed sorghum seeds at 26 Ϯ 1 °C, 60 Ϯ 5% relative humidity, and a 14:10-h light:dark photo period. The fat body and other insect tissue homogenates from ligated or unligated larvae were prepared as published earlier [22] and used after protein estimation in an aliquot of the homogenate [23]. The reaction mixture contained 150 mM sodium acetate buffer (pH 5.0) and 20 ␮g of tissue homogenate proteins. It was incubated at 37 °C for 10 min to exclude glucose-6phosphatase activity [25]. The reaction was initiated by the addition of 5 ␮mol of substrate, p-nitrophenyl bisodium phosphate (Sigma) to the assay mixture followed by incubation for 1 h at 37 °C.

The abbreviations used are

RESULTS

DISCUSSION