title: Site-specific glycosylation at Asn-292 in ovalbumin is essential to efficient secretion in yeast.
abstract: Chicken ovalbumin (OVA) exists as mono-N-glycosylated form with a carbohydrate chain on Asn-292 in egg white, despite the possession of two potential N-glycosylation sites. To investigate the roles of N-glycosylation of OVA, we constructed a series of N-glycosylation mutants deleted N-glycosylation site and compared the secretion level of the mutants in Pichia pastoris. N292Q and N292/311Q mutants resulted in greater lowering of the secretion level as compared with wild-type, whereas N311Q mutant was secreted in approximately equal amounts to wild-type. However, secretion of wild-type and N311Q mutant was inhibited completely by tunicamycin treatment. All the N-glycosylation mutants have been expressed in the cells, as well as wild-type. Circular dichroism and fluorescence spectra of secreted N311Q mutant were almost identical to those of wild-type, while those of N292Q and N292/311Q mutants were different from wild-type; and, N292Q and N292/311Q mutants showed considerably lower denaturation temperature than wild-type. The results indicate that N-glycosylation at Asn-292 of OVA is required for the folding and secretion.
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Ito
authors.forename: Akiko
authors.initials: A
authors.lastname: Seri
authors.forename: Fukiko
authors.initials: F
authors.lastname: Kimura
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Matsudomi
mesh_terms: D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D006031:Glycosylation; D010047:Ovalbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D030161:Avian Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D010047:Ovalbumin