Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

Abstract The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys 10 and Cys 17 , and Cys 151 and Cys 195 . Of the two potential N-glycosylation sites in JHBP, Asn 4 , and Asn 94 , only Asn 94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris .