Spiro R G, Zhu Q, Bhoyroo V, Söling H D
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts, USA.
J Biol Chem. 1996 May 10;271(19):11588-94. doi: 10.1074/jbc.271.19.11588.
Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.
通过免疫化学和序列分析鉴定出，钙网蛋白是先前在通过含Glcα1→3Man基质进行色谱分离得到的大鼠肝脏高尔基体甘露糖苷酶制剂中观察到的多肽双峰中分子量较高（60 kDa）的组分。对这种糖配体的亲和力与甘露糖苷酶的亲和力相似，并且在纯化的大鼠肝脏钙网蛋白中也观察到这种亲和力，这表明这种伴侣蛋白具有类凝集素结合特性。用固定化钙网蛋白和一系列源自N-连接碳水化合物单元的放射性标记寡糖进行的研究表明，与该蛋白的相互作用仅限于单葡萄糖基化的多聚甘露糖组分。尽管与Glc1Man9GlcNAc发生了最佳结合，但在将多聚甘露糖部分依次修剪至Glc1Man5GlcNAc阶段后，仍保留了与钙网蛋白的大量相互作用。然而，寡糖核心的α1→6-甘露糖分支点似乎对于识别至关重要，因为Glc1Man4GlcNAc不与钙网蛋白相互作用。碳水化合物-肽连接区对结合没有明显影响，因为N-糖苷键连接的单葡萄糖基化寡糖与伴侣蛋白的相互作用程度与其未结合状态相同。固定化钙网蛋白被证明是从复杂的加工中间体混合物中筛选出单葡萄糖基化多聚甘露糖寡糖或糖肽的高效工具。钙网蛋白和甘露糖苷酶从高尔基体组分中以相当的量共纯化，并且伴侣蛋白和加工酶的糖特异性惊人地相似，这提示了一个初步模型，即在内质网后区域通过去除葡萄糖来解离钙网蛋白-糖蛋白复合物；在这个方案中，去糖基化将由甘露糖苷酶而不是葡糖苷酶II的作用引起。
