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Journal of Biochemistry Advance Access originally published online on December 13, 2006
Journal of Biochemistry 2007 141(2):193-199; doi:10.1093/jb/mvm021
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© 2006 The Japanese Biochemical Society.

Site-specific Glycosylation at Asn-292 in Ovalbumin is Essential to Efficient Secretion in Yeast

Kazunari Ito, Akiko Seri, Fukiko Kimura and Naotoshi Matsudomi*

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan

*To whom correspondence should be addressed. Tel: +81-83-933-5853, Fax: +81-83-933-5820, E-mail: naotoshi{at}yamaguchi-u.ac.jp

Received November 9, 2006; Accepted November 28, 2006


  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.

Key Words: folding, N-glycosylation, ovalbumin, Pichia pastoris, secretion

Abbreviations: OVA, ovalbumin; Endo H, endoglycosidase H; PNGase F, peptide:N-glycanase F; CD, circular dichroism


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