α1-antitrypsin (AAT) is a serine protease inhibitor synthesized in hepatocytes and protects the lung from damage by neutrophil elastase. AAT gene mutations result in AAT deficiency (AATD), which leads to lung and liver diseases. The AAT Z variant forms polymer within the endoplasmic reticulum (ER) of hepatocytes and results in reduction in AAT secretion and severe disease. Previous studies demonstrated a secretion defect of AAT in LMAN1 deficient cells, and mild decreases in AAT levels in male LMAN1 and MCFD2 deficient mice. LMAN1 is a transmembrane lectin that forms a complex with a small soluble protein MCFD2. The LMAN1–MCFD2 protein complex cycles between the ER and the Golgi. Here, we report that LMAN1 and MCFD2 knockout (KO) HepG2 and HEK293T cells display reduced AAT secretion and elevated intracellular AAT levels due to a delayed ER-to-Golgi transport of AAT. Secretion defects in KO cells were rescued by wild-type LMAN1 or MCFD2, but not by mutant proteins. Elimination of the second glycosylation site of AAT abolished LMAN1 dependent secretion. Co-immunoprecipitation experiment in MCFD2 KO cells suggested that AAT interaction with LMAN1 is independent of MCFD2. Furthermore, our results suggest that secretion of the Z variant, both monomers and polymers, is also LMAN1-dependent. Results provide direct evidence supporting that the LMAN1–MCFD2 complex is a cargo receptor for the ER-to-Golgi transport of AAT and that interactions of LMAN1 with an N-glycan of AAT is critical for this process. These results have implications in production of recombinant AAT and in developing treatments for AATD patients.

中文翻译：

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LMAN1-MCFD2 复合物是 ER-Golgi 转运 α1-抗胰蛋白酶的货物受体

α1-抗胰蛋白酶 (AAT) 是一种在肝细胞中合成的丝氨酸蛋白酶抑制剂，可保护肺免受中性粒细胞弹性蛋白酶的损伤。AAT 基因突变导致 AAT 缺乏症 (AATD)，从而导致肺和肝病。AAT Z 变体在肝细胞的内质网 (ER) 内形成聚合物，导致 AAT 分泌减少和严重疾病。先前的研究表明，LMAN1 缺陷细胞中 AAT 的分泌缺陷，雄性 LMAN1 和 MCFD2 缺陷小鼠的 AAT 水平轻度下降。LMAN1 是一种跨膜凝集素，可与小的可溶性蛋白 MCFD2 形成复合物。LMAN1-MCFD2 蛋白复合物在 ER 和高尔基体之间循环。这里，我们报告 LMAN1 和 MCFD2 敲除 (KO) HepG2 和 HEK293T 细胞显示 AAT 分泌减少和细胞内 AAT 水平升高，这是由于 AAT 延迟 ER 到高尔基体的运输。KO 细胞中的分泌缺陷被野生型 LMAN1 或 MCFD2 拯救，但不是由突变蛋白拯救。消除 AAT 的第二个糖基化位点消除了 LMAN1 依赖性分泌。MCFD2 KO 细胞中的免疫共沉淀实验表明 AAT 与 LMAN1 的相互作用不依赖于 MCFD2。此外，我们的结果表明，单体和聚合物的 Z 变体的分泌也依赖于 LMAN1。结果提供了直接证据，支持 LMAN1-MCFD2 复合物是 AAT 的 ER 到高尔基体转运的货物受体，并且 LMAN1 与 AAT 的 N-聚糖的相互作用对该过程至关重要。