Distinct patterns of folding and interactions with calnexin and calreticulin in human class I MHC proteins with altered N-glycosylation.
Distinct patterns of folding and interactions with calnexin and calreticulin in human class I MHC proteins with altered N-glycosylation.
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DOI：
10.4049/jimmunol.160.2.831
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发表时间：
1998-01
影响因子：
4.4
通讯作者：
Q. Zhang;R. Salter
中科院分区：
文献类型：
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作者：
Q. Zhang;R. Salter
钙连接蛋白是一种凝集素样分子伴侣，在I类MHC分子合成后不久与其结合，保留未组装的重链并帮助其折叠。在内质网中与β 2-微球蛋白（β 2 m）结合后，大部分人I类分子从钙连接蛋白释放，而小鼠I类分子则不释放。我们询问在人I类分子A*0201的176位（存在于小鼠中的位点）添加第二个N-聚糖是否会影响其与钙连接蛋白的结合。当转染到CIR细胞中时，在位置86和176处具有N-聚糖的176 dg突变体表现出与钙连接蛋白的结合增加，在与β 2 m缔合之前和之后均可检测到，并且相对于在位置86处具有单个N-聚糖的野生型蛋白，与钙网蛋白和TAP的相互作用减少。突变体的细胞表面水平相对于野生型仅略微降低，表明蛋白质没有错误折叠或结构上发生严重改变。突变分子的亚群保留在内质网中，并且令人惊讶的是，这些分子与w 6/32反应，其识别存在于有运输能力的I类HLA复合物上的表位。转染到Daudi细胞中证明176 dg在不存在β 2 m的情况下与w 6/32反应，表明Ab表位可以通过钙连接蛋白的结合来诱导。这些数据可以解释先前注意到的小鼠和人类I类MHC蛋白质之间的差异，并证明蛋白质内的N-寡糖的位置可以影响它们的折叠和与分子伴侣如钙连接蛋白和钙网蛋白的相互作用。
Calnexin is a lectin-like chaperone that binds to class I MHC molecules soon after their synthesis, retaining unassembled heavy chains and also assisting their folding. Following association with beta2-microglobulin (beta2m) in the endoplasmic reticulum, a large proportion of human class I molecules release from calnexin, whereas mouse class I molecules do not. We asked whether addition of a second N-glycan to the human class I molecule A*0201 at position 176, a site present in mouse, would affect its binding to calnexin. The 176dg mutant with N-glycans at positions 86 and 176, when transfected into CIR cells, demonstrated increased binding to calnexin, detectable both before and after association with beta2m, and reduced interaction with calreticulin and TAP relative to wild-type protein bearing a single N-glycan at position 86. Cell surface levels of the mutant were decreased only slightly relative to the wild type, suggesting that the protein is not misfolded or grossly altered structurally. A subpopulation of mutant molecules was retained in the endoplasmic reticulum, and surprisingly, these molecules reacted with w6/32, which recognizes an epitope present on transport-competent class I HLA complexes. Transfection into Daudi cells demonstrated that 176dg reacts with w6/32 in the absence of beta2m, suggesting that the Ab epitope can be induced by binding of calnexin. These data may explain previously noted differences between mouse and human class I MHC proteins and demonstrate that the location of N-oligosaccharides within proteins can influence their folding and interactions with chaperones such as calnexin and calreticulin.