Key Disulfide Bonds in an Insect Hormone Binding Protein: cDNA Cloning of a Juvenile Hormone Binding Protein of Heliothis virescens and Ligand Binding by Native and Mutant Forms

Abstract

The hemolymph juvenile hormone binding protein (JHBP) from the early fifth instar larvae of Heliothis virescens (Lepidoptera, Noctuidae) has been purified, and three cDNA clones for this protein have been isolated from a fat body cDNA library constructed in bacteriophage lambda ZAP XR. The deduced amino acid sequence of the full-length clone predicts a mature protein consisting of 224 residues, a molecular mass of 24,976 Da, and a pI of 5.29. Comparison of the amino acid sequence to that of the previously described JHBP from Manduca sexta shows 51% overall identity with highly conserved N- and C-terminal regions. One of the three clones bound photoactivatable analogs of juvenile hormones with much lower affinity than the other two. This clone had Phe150 in place of the expected Cys150 conserved in other JHBP clones. The F150C mutant of this clone regained native binding affinity. For native Hvir-JHBP, the affinity for [3H]JH I was lower under reducing conditions (87 nM) relative to a 40 nM affinity under nonreducing conditions. The importance of pairs of Cys residues was addressed by preparing Cys to Ala mutants at each site. Expressed proteins were tested for binding affinity by photoaffinity labeling with tritium-labeled JH analogs and by binding assays using (10R,11S)-[3H]JH I. Curiously, the C150A mutant retained full activity, implying that the aberrant C150F was dysfunctional due to steric hindrance rather than to a missing disulfide linkage. Likewise, C29A and C194A had binding affinities unchanged from that of the full-length wild-type clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of male‐specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L.

“…There is also no tryptophan residue in those proteins except only one residue in B. mori JHBP. The shift of Mr observed in G. mellonella MSP was also observed in JHBPs from SDS-PAGE (Touhara et al, 1993;Kurata et al, 1994;Wojtasek and Prestwich, 1995;Vermunt et al, 2001;Parkitna et al, 2002). Based on these facts, MSP of G. mellonella could be an outer member of the JHBP family.…”

“…1987; Lerro and Prestwich, 1990;Wojtasek and Prestwich, 1995;Park and Kim, 1996;Vermunt et al, 2001;Parkitna et al, 2002). Four conserved cysteins found in JHBPs were also found in MSP.…”

“…Four conserved cysteins found in JHBPs were also found in MSP. These cysteins were known to be crucial in the tertiary structure and the JH binding function by making two disulfide bonds in H. virescens JHBP (Wojtasek and Prestwich, 1995). There is also no tryptophan residue in those proteins except only one residue in B. mori JHBP.…”

Evidence for Glycosylation of the Juvenile‐Hormone‐Binding Protein from Galleria mellonella Hemolymph

“…Unfortunately, the complete amino acid sequence of the G. mellonellu JHBP is not known, but the known sequences of two other JHBP, from M. sexta and H. virescens hemolymph, show the presence of one potential glycosylation site, at Asn56 in M. sextu and Asnl9 in H. virescens JHBP (Wojtasek and Prestwich, 1995). Interestingly, the Asnl9 residue was not detected during sequencing of the Nterminal peptide of H. virescens JHBP by Edman degradation (Wojtasek and Prestwich, 1995), which suggests glycosylation of this residue. The authors did not comment on this result and did not consider the possibility of glycosylation of this residue, presumably because early studies on the closely related M. sexta JHBP did not indicate a detectable content of carbohydrates (Goodman et al, 1978).…”

The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior

“…The juvenile hormone-binding proteins from other insects, to which Takeout is related, are synthesized in the fat body and secreted into the hemolymph, where they are thought to carry juvenile hormone or other small lipophilic ligands to target cells (Nowock et al 1975;Glinka et al 1995;Wojtasek and Prestwich 1995). It is not known whether juvenile hormone plays any role in Drosophila male courtship behavior, but it has been shown that juvenile hormone stimulates the synthesis of accessory gland proteins (Herndon et al 1997).…”