Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation, OH 44195, USA.
Blood. 2010 Feb 4;115(5):1081-7. doi: 10.1182/blood-2009-09-241877. Epub 2009 Dec 9.
Combined deficiency of factor V and factor VIII (F5F8D) is a bleeding disorder caused by mutations in either LMAN1 or MCFD2. LMAN1 (ERGIC-53) and MCFD2 form a Ca(2+)-dependent cargo receptor that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment for efficient transport of FV/FVIII from the ER to the Golgi. Here we show that the C-terminal EF-hand domains are both necessary and sufficient for MCFD2 to interact with LMAN1. MCFD2 with a deletion of the entire N-terminal non-EF hand region still retains the LMAN1-binding function. Deletions that disrupt core structure of the EF-hand domains abolish LMAN1 binding. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF-hand domains suggest that Ca(2+)-induced folding is important for LMAN1 interaction. The EF-hand domains also mediate the interaction with FV and FVIII. However, mutations in MCFD2 that disrupt the tertiary structure and abolish LMAN1 binding still retain the FV/FVIII binding activities, suggesting that this interaction is independent of Ca(2+)-induced folding of the protein. Our results suggest that the EF-hand domains of MCFD2 contain separate binding sites for LMAN1 and FV/FVIII that are essential for cargo receptor formation and cargo loading in the ER.
因子 V 和因子 VIII 联合缺乏症（F5F8D）是一种由 LMAN1 或 MCFD2 突变引起的出血性疾病。LMAN1（ERGIC-53）和 MCFD2 形成一个依赖 Ca(2+)的货物受体，在 ER 和 ER-Golgi 中间隔室之间循环，以有效地将 FV/FVIII 从 ER 转运到 Golgi。在这里，我们表明 C 端 EF 手结构域对于 MCFD2 与 LMAN1 相互作用都是必要和充分的。缺失整个 N 端非 EF 手区域的 MCFD2 仍然保留与 LMAN1 结合的功能。破坏 EF 手结构域核心结构的缺失会破坏 LMAN1 的结合。针对 EF 手结构域不同结构元件的错义突变的圆二色性光谱研究表明，Ca(2+)诱导的折叠对于 LMAN1 相互作用很重要。EF 手结构域还介导与 FV 和 FVIII 的相互作用。然而，破坏三级结构并废除 LMAN1 结合的 MCFD2 突变仍然保留 FV/FVIII 的结合活性，这表明这种相互作用独立于蛋白的 Ca(2+)诱导折叠。我们的研究结果表明，MCFD2 的 EF 手结构域包含 LMAN1 和 FV/FVIII 的独立结合位点，对于货物受体形成和 ER 中的货物装载至关重要。
