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

Abstract

The juvenile-hormone-binding protein (JHBP) from Galleria mellonella hemolymph, which is a member of the high-affinity/low-molecular-mass group of JHBP proteins, was found to be glycosylated. Glycosylation was confirmed by the following evidence. Carbohydrate gas-liquid chromatography analysis of the purified JHBP preparations showed the presence of a low amount of sugars (Man and GlcNAc were the major components). The JHBP electrophoretic band blotted onto nitrocellulose was stained with GlycoTrack (a reagent kit used for the detection of protein glycosylation) and showed strong binding of concanavalin A (ConA). JHBP was fractionated on a ConA-Sepharose 4B column into ConA-bound (strongly stained with ConA) and ConA-unbound (hardly stained with ConA) portions. Both fractions showed juvenile-hormone-binding activity and were glycosylated, as revealed by staining both of them with GlycoTrack. Electrospray-ionization mass spectrometry of JHBP suggested the presence of a small amount of presumably nonglycosylated protein (24 988 Da) and five glycoforms, two of which (containing Man,GlcNAc, or Man,Fuc,GlcNAc, chain) were not bound or were weakly bound to ConA, and three (with Man,GlcNAc,, Man,Fuc, GlcNAc,, or Man,GlcNAc, chain) were present in the fraction strongly bound to ConA. In conclusion, the monosugar composition, GlycoTrack staining, ConA-binding properties and molecular mass analyses of JHBP supplied convincing evidence for its glycosylation and some information on the character of the oligosaccharide chains.

Insect Juvenile Hormone Binding Protein Shows Ancestral Fold Present in Human Lipid-Binding Proteins

“…The central part of the β-sheet is five-stranded, while near the ends of the helical axis the wrap has only four β-strands, because the edges of the β-sheet structure are disrupted by additional, shorter helices. One such helix (α1) is present near the very N-terminus of the sequence (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), which is spatially adjacent to the C-terminus of helix α4. At the opposite end, two helices (α2, α3) form a right-angle motif, which after another perpendicular kink leads directly to α4.…”

“…16 The mature protein is variously glycosylated at a single site, but the mass of the sugar component does not exceed 10% 17,18 of the protein mass. There are no Met or Trp residues in the sequence and the Cys residues are paired to form two disulfide bridges (Cys10-Cys17, Cys151-Cys195).…”

The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis

“…Putative post‐translational modifications, such as glycosylations, may contribute to a higher M r . In another lepidopteran, G. mellonella, hJHBP was found to be glycosylated (Duk et al. , 1996).…”

Electrochemical impedance spectroscopy for the study of juvenile hormones - recombinant protein interactions

“…This protein has been purified to homogeneity and characterized (15). It is a single chain basic glycoprotein (pI = 8.1), which possess one binding site for JH (14,15,16). In SDS-PAGE this protein exhibits a relative molecular mass close to 32 kDa (15,12).…”