IP Endocrinology

Juvenile hormone esterase binding proteins in Manduca sexta

M. Shanmugavelu 1, A. R. Baytan 2, J. D. Chesnut 2 & B. C. Bonning 1 (Home page)

1 Dept. Entomology, Iowa State Univ., 418 Science II, Ames, IA 50011, USA; 2 Invitrogen, 1600 Faraday Ave., Carlsbad, CA 92008, USA

Juvenile hormone esterase (JHE) hydrolyses juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating JHE is synthesized by the fat body and epidermis, and removed from hemolymph by pericardial cells for lysosomal degradation. For analyses of binding proteins in the tobacco hornworm Manduca sexta L., baculovirus expressed, recombinant JHE was purified by ion exchange chromatography and biotinylated, for use in ligand blot. Five putative JHE binding proteins of 29, 50, 75, 125 and 240 kDa were identified in both fat body and pericardial cell tissues. All binding proteins were present from second through fifth instar larvae of M. sexta. On narrow-range isoelectric focusing, the 29 kDa binding protein separated into three species with isoelectric points of 6.48, 6.63 and 6.80. In order to identify proteins involved in the degradation of JHE, a pericardial cell cDNA phage display library derived from M. sexta was constructed and screened for proteins that bind JHE. A 732 bp cDNA encoding a novel 29 kDa protein, P29, was isolated. Western and northern analyses indicate that P29 is present in both pericardial cell and fat body tissue and is present in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant JHE taken up by pericardial cells, and also native M. sexta JHE in fat body tissue. P29 bound JHE more strongly that it did a mutant form of the enzyme JHE-KK, with mutations that perturb lysosomal targeting. Future research will address the roles of JHE binding proteins in synthesis and export and/or uptake and degradation of JHE by the pericardial cells and fat body of M. sexta.

Index terms: protein trafficking, lysosome, pericardial cells, fat body.

Copyright: The copyrights of this original work belong to the authors (see right-most box in title table). This abstract appeared in Session 13 – INSECT PHISIOLOGY, NEUROSCIENCES, IMMUNITY AND CELL BIOLOGY Symposium and Poster Session, ABSTRACT BOOK II – XXI-International Congress of Entomology, Brazil, August 20-26, 2000.