Evidence for glycosylation of the juvenile-hormone-binding protein from Galleria mellonella hemolymph.
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 (24988 Da) and five glycoforms, two of which (containing Man2GlcNAc, or Man2Fuc1GlcNAc2 chain) were not bound or were weakly bound to ConA, and three (with Man3GlcNAc2, Man5Fuc1GlcNAc2, or Man5GlcNAc2, 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.
Duk
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Zaklad Immunochemii, Instytut Immunologii i Terapii Doswiadczalnej PAN im. Ludwika Hirszfelda, Wroclaw, Polska.
Krotkiewski
H
H
Forest
E
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Rodriguez Parkitna
J M
JM
Kochman
M
M
Lisowska
E
E
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry*; D013058:Mass Spectrometry; D009036:Moths / Q000737:chemistry*
D002352:Carrier Proteins; D006023:Glycoproteins; D019476:Insect Proteins; C035606:juvenile hormone-binding protein, insect; D003208:Concanavalin A; D006026:Glycoside Hydrolases