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Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site‐specific glycosylation in yeast
Author(s) -
He Jianwei,
Song Youtao,
Ueyama Nobuhiro,
Saito Akira,
Azakami Hiroyuki,
Kato Akio
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051753306
Subject(s) - glycosylation , cystatin , chemistry , biochemistry , n linked glycosylation , pichia pastoris , yeast , amyloid (mycology) , mutant , cystatin c , glycoprotein , recombinant dna , gene , glycan , inorganic chemistry , renal function
To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N ‐linked glycosylation (Asn 106 ‐Ile 108 → Asn 106 ‐Thr 108 ) 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.