Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity.
Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity.
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DOI：
10.1111/j.1742-4658.2011.08351.x
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发表时间：
2011-11
期刊：
影响因子：
--
通讯作者：
Sahin-Tóth M
中科院分区：
文献类型：
--
作者：
Bence M;Sahin-Tóth M
人胰凝乳酶C(CTRC)通过降解减轻胰蛋白酶原的过早激活，在胰腺中起到保护作用。取消CTRC活性或分泌的突变会增加慢性胰腺炎的风险。本研究的目的是确定人CTRC是否经历天冬酰胺连接(N连接)的糖基化，并研究这种修饰在CTRC折叠和功能中的作用。我们通过将人CTRC中的ASN残基单独或联合突变为Ser，消除了人CTRC中潜在的糖基化位点(Asn-Xaa-Ser/Thr)，在HEK 293T细胞中表达了CTRC突变体，并用PNGase F和Endo H双酶切鉴定了它们的糖基化状态。我们发现人CTRC在Asn52上含有一个N-连接的糖链。Asn52(N52S)突变消除N-糖基化使HEK 293T细胞分泌CTRC减少约10倍，但不影响CTRC活性或抑制物结合。N52S CTRC突变体的过表达在AR42J腺泡细胞中引起内质网应激，表明人CTRC的折叠需要N-糖基化。尽管Asn52具有重要的作用，但它在其他哺乳动物的CTRC同源基因中的保守性很差，包括在Asn90上单糖基化的大鼠。在一个非糖基化的人CTRC突变体中引入Asn90位点后，完全恢复了糖基化，但仅部分修复了分泌缺陷。我们的结论是，人CTRC的N-连接糖基化是有效折叠和分泌所必需的，然而，N-连接的糖链对酶活性或抑制物结合并不重要。N-连接的糖链的位置对最佳折叠至关重要，在其他高度同源的哺乳动物CTRC序列中，它可能会有所不同。
Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC by mutating the Asn residues to Ser individually or in combination, expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 (N52S) reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells, indicating that N-glycosylation is required for folding of human CTRC. Despite its important role, Asn52 is poorly conserved in other mammalian CTRC orthologs, including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation but only partially rescued the secretion defect. We conclude that N-linked glycosylation of human CTRC is required for efficient folding and secretion, however, the N-linked glycan is unimportant for enzyme activity or inhibitor binding. The position of the N-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences.
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影响因子：
6.5
作者：
Miller, GC;Long, CJ;Rader, DJ
通讯作者：
Rader, DJ
影响因子：
4.8
作者：
Nemoda, Z;Sahin-Tóth, M
通讯作者：
Sahin-Tóth, M
影响因子：
4.8
作者：
Szabo, Andras;Heja, David;Pal, Gabor
通讯作者：
Pal, Gabor
DOI：
10.1083/jcb.200808124
发表时间：
2009-03-23
期刊：
The Journal of cell biology
影响因子：
--
作者：
Glozman R;Okiyoneda T;Mulvihill CM;Rini JM;Barriere H;Lukacs GL
通讯作者：
Lukacs GL
影响因子：
24.5
作者：
Szmola R;Sahin-Tóth M
通讯作者：
Sahin-Tóth M