Rodan A R, Simons J F, Trombetta E S, Helenius A
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520-8002, USA.
EMBO J. 1996 Dec 16;15(24):6921-30.
Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.
钙连蛋白和钙网蛋白是凝集素样分子伴侣，可促进内质网中新合成糖蛋白的折叠和组装。虽然它们与底物单葡萄糖基化N-连接寡糖相互作用已得到充分证实，但也有人提出它们还与多肽部分相互作用。为了验证这一观点，在微粒体存在的情况下翻译了糖基化形式的牛胰腺核糖核酸酶（RNase），并监测了其折叠以及与钙连蛋白和钙网蛋白的结合情况。当在微摩尔浓度的脱氧野尻霉素存在下表达带有两个N-连接聚糖时，发现这种小的可溶性蛋白能牢固地结合钙连蛋白和钙网蛋白。寡糖对于结合是必需的，但RNase是否折叠并无影响。这表明与其他分子伴侣不同，钙连蛋白和钙网蛋白不是根据折叠状态来选择底物的。此外，使用肽N-糖苷酶F酶促去除寡糖链或通过内质网葡糖苷酶II去除葡萄糖会导致复合物解离。这表明凝集素样相互作用而非蛋白质-蛋白质相互作用在稳定RNase-钙连蛋白/钙网蛋白复合物中起核心作用。
