title: N-linked glycosylation of G. mellonella juvenile hormone binding protein - comparison of recombinant mutants expressed in P. pastoris cells with native protein.
abstract: Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn(94)) of the two potential N-linked glycosylation sites (Asn(4) and Asn(94)). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn(94) reduces the level of protein secretion whereas mutation at the Asn(4) site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.
authors.affiliation: Department of Biochemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Winiarska
authors.forename: Angela
authors.initials: A
authors.lastname: Dwornik
authors.forename: Janusz
authors.initials: J
authors.lastname: Dębski
authors.forename: Krystyna
authors.initials: K
authors.lastname: Grzelak
authors.forename: Dominika
authors.initials: D
authors.lastname: Bystranowska
authors.forename: Marta
authors.initials: M
authors.lastname: Zalewska
authors.forename: Michał
authors.initials: M
authors.lastname: Dadlez
authors.forename: Andrzej
authors.initials: A
authors.lastname: Ożyhar
authors.forename: Marian
authors.initials: M
authors.lastname: Kochman
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007605:Juvenile Hormones / Q000378:metabolism*; D009036:Moths / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D002352:Carrier Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; D011994:Recombinant Proteins