Department of Biochemistry, Wakayama Medical University, Wakayama, Japan.
Synthetic Cellular Chemistry Laboratory, RIKEN, Saitama, Japan.
Methods Mol Biol. 2020;2132:295-308. doi: 10.1007/978-1-0716-0430-4_29.
Calnexin (CNX) and calreticulin (CRT) are ER-resident lectin-like molecular chaperones involved in the quality control of secretory or membrane glycoproteins. They can exert molecular chaperone functions via specific binding to the early processing intermediates of GlcManGlcNAc oligosaccharides of N-glycoproteins. CNX and CRT have similar N-terminal luminal domains and share the same jelly roll tertiary structure as legume lectins. In addition to the lectin-like interactions, CNX and CRT also suppress the aggregation of non-glycosylated substrates through interaction with hydrophobic peptide parts, suggesting a general chaperone function in glycan-dependent and glycan-independent manners. This chapter describes the isolation and purification of CRT produced in a bacterial expression system. We also introduce in vitro assays to estimate the molecular chaperone functions of CRT via the interaction with monoglucosylated N-glycans using Jack bean α-mannosidase as a target substrate. These assays are valuable in assessing quality control events related to the CNX/CRT chaperone cycle and lectin functions.
钙连蛋白（CNX）和钙网蛋白（CRT）是内质网驻留的凝集素样分子伴侣，参与分泌或膜糖蛋白的质量控制。它们可以通过与 N-糖蛋白 GlcManGlcNAc 寡糖的早期加工中间体特异性结合来发挥分子伴侣功能。CNX 和 CRT 具有相似的 N 端腔域，并与豆科植物凝集素有相同的果冻卷三级结构。除了凝集素样相互作用外，CNX 和 CRT 还通过与疏水性肽部分的相互作用抑制非糖基化底物的聚集，表明它们以糖依赖性和非糖依赖性方式具有普遍的伴侣功能。本章描述了在细菌表达系统中产生的 CRT 的分离和纯化。我们还介绍了体外测定，以使用刺槐豆α-甘露糖苷酶作为靶底物，通过与单葡萄糖化 N-聚糖的相互作用来评估 CRT 的分子伴侣功能。这些测定对于评估与 CNX/CRT 伴侣循环和凝集素功能相关的质量控制事件非常有价值。
