EVIDENCE FOR GLYCOSYLATION OF THE JUVENILE-HORMONE-BINDING PROTEIN FROM GALLERIA-MELLONELLA HEMOLYMPH

The juvenile-hormone-binding protein (JHBP) from Galleria mellonella h emolymph, which is a member of the high-affinity/low-molecular-mass gr oup of JHBP proteins, was found to be glycosylated. Glycosylation was confirmed by the following evidence. Carbohydrate gas-liquid chromatog raphy analysis of the purified JHBP preparations showed the presence o f a low amount of sugars (Man and GlcNAc were the major components). T he JHBP electrophoretic band blotted onto nitrocellulose was stained w ith GlycoTrack (a reagent kit used for the detection of protein glycos ylation) and showed strong binding of concanavalin A (ConA). JHBP was fractionated on a ConA-Sepharose 4B column into ConA-bound (strongly s tained with ConA) and ConA-unbound (hardly stained with ConA) portions . Both fractions showed juvenile-hormone-binding activity and were gly cosylated, as revealed by staining both of them with GlycoTrack. Elect rospray-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(2)GlcNAc(2) or Man(2)Fuc (1)GlcNAc(2) chain) were not bound or were weakly bound to ConA, and t hree (with Man(3)GlcNAc(2), Man(3)Fuc(1)GlcNAc(2), or Man(5)GlcNAc(2) chain) were present in the fraction strongly bound to ConA. In conclus ion, the monosugar composition, GlycoTrack staining, ConA-binding prop erties and molecular mass analyses of JHBP Supplied convincing evidenc e for its glycosylation and some information on the character of the o ligosaccharide chains.