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Protein Science (2006), 15:213-222. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast

Jianwei He1, Youtao Song2, Nobuhiro Ueyama1, Akira Saito3, Hiroyuki Azakami1 and Akio Kato1

1 Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan
2 Department of Life Science, Liaoning Universtity, Shenyang, 110036, China
3 Institute of Cellular and Molecular Medicine, University of California, San Diego, California 92093, USA

(RECEIVED August 7, 2005; FINAL REVISION October 10, 2005; ACCEPTED October 27, 2005)

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 -> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.

Keywords: amyloidogenic chicken cystatin; amyloid fibrils; Pichia pastoris; glycosylation modification; quality control

Abbreviations: HCCAA, human cystatin C amyloid angiopathy • hcC, human cystatin C • cC, chicken cystatin • CD, circular dichroism • ER, endoplasmic reticulum • PAGE, polyacrylamide gel electrophoresis • RT-PCR, reverse transcriptase-polymerase chain reaction

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051753306.

Reprint requests to: Akio Kato, Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan; e-mail: akiokato{at}yamaguchi-u.ac.jp; fax: +81-83-933-5820.


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