Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast.
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.
He
Jianwei
J
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan.
Song
Youtao
Y
Ueyama
Nobuhiro
N
Saito
Akira
A
Azakami
Hiroyuki
H
Kato
Akio
A
D000682:Amyloid / Q000378:metabolism* / Q000648:ultrastructure; D000686:Amyloidosis / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
D000682:Amyloid; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; C100158:cystatin, egg-white