He Jianwei, Song Youtao, Ueyama Nobuhiro, Saito Akira, Azakami Hiroyuki, Kato Akio
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan.
Protein Sci. 2006 Feb;15(2):213-22. doi: 10.1110/ps.051753306.
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.
为了研究糖基化对淀粉样变鸡半胱氨酸蛋白酶原纤维形成能力的作用，通过定点诱变将N - 糖基化的共有序列（天冬酰胺106 - 异亮氨酸108→天冬酰胺106 - 苏氨酸108）引入野生型和淀粉样变鸡半胱氨酸蛋白酶原中，构建鸡半胱氨酸蛋白酶原的糖基化形式。野生型和淀粉样变突变体I66Q半胱氨酸蛋白酶原的糖基化和非糖基化形式均在酵母毕赤酵母转化体的培养基中表达并分泌。对不溶性聚集体的量、二级结构和原纤维形成能力的比较表明，N - 糖基化可以防止酵母细胞中分泌的淀粉样变鸡半胱氨酸蛋白酶原形成淀粉样原纤维，而不影响其抑制活性。进一步的研究表明这种糖基化可以抑制半胱氨酸蛋白酶原二聚体的形成。因此，我们的数据强烈表明，防止淀粉样变半胱氨酸蛋白酶原纤维形成的机制可能是通过106位糖基化链抑制淀粉样变半胱氨酸蛋白酶原的三维结构域交换二聚体和寡聚体的形成来实现的。
