Takeda Pharmaceuticals, Cambridge, MA, 02140, USA.
Takeda Pharmaceuticals, Cambridge, MA, 02140, USA.
Protein Expr Purif. 2020 Jun;170:105589. doi: 10.1016/j.pep.2020.105589. Epub 2020 Feb 3.
The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization.
阳离子非依赖型甘露糖-6-磷酸受体（CI-M6PR，也称为胰岛素样生长因子 II 受体或 IGFIIR）是一种膜蛋白，在通过甘露糖-6-磷酸（M6P）结合域将溶酶体酸性水解酶运输到溶酶体中方面发挥核心作用。为了维持细胞代谢/分解代谢的平衡，新合成的溶酶体酸性水解酶需要与 M6PR 结合以进行转运。细胞分泌的酸性水解酶也可以通过存在于细胞膜上的 M6PR 内化，并被运输到溶酶体，这一特性使酶替代疗法能够治疗几种溶酶体贮积症。因此，对这种受体进行彻底的表征对于开发基于溶酶体酶的治疗方法至关重要，这些治疗方法利用 M6PR 将药物递送到溶酶体。然而，M6PR 的细胞外结构域（ECD）非常复杂，包含 15 个甘露糖受体同源（MRH）结构域。此外，受体可以在膜上发生同源二聚化，这使其表征具有挑战性。在这项研究中，利用最初为 hM6PR 细胞摄取测定而建立的新型人 M6PR（hM6PR）过表达细胞系来生产 hM6PR-ECD，并开发了一种新型小分子模拟物（氨苯基-M6P）亲和树脂用于 M6PR-ECD 的纯化。亲和纯化的 hM6PR-ECD 是单体的，包含 14 个完整的 MRH 结构域（1-14）和部分 MRH 结构域 15，并成功用于基于 ELISA 和表面等离子体共振的结合测定中，以证明其配体结合功能，使其适合评估利用 M6PR 进行细胞内化的生物疗法。
