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.

中文翻译：

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人甘露糖6-磷酸酯受体的表达，纯化和表征-利用甘露糖6-磷酸酯部分的小分子仿生剂来自稳定细胞系的细胞外结构域。

阳离子非依赖性甘露糖6磷酸受体（CI-M6PR，又名胰岛素样生长因子II受体或IGFIIR）是一种膜蛋白，在通过6磷酸甘露糖将溶酶体酸水解酶运输到溶酶体中起着核心作用（M6P）绑定域。为了维持细胞代谢/分解代谢的稳态，需要新合成的溶酶体酸水解酶与M6PR结合以便转运。细胞分泌的酸性水解酶也可以通过驻留在细胞膜上的M6PR内在化，然后转运到溶酶体中，这一功能使酶替代疗法能够治疗多种溶酶体贮积症。因此，对该受体的彻底表征对于开发基于溶酶体酶的疗法至关重要，该疗法利用M6PR将药物递送至溶酶体。然而，M6PR的细胞外结构域（ECD）非常复杂，包含15个甘露糖受体同源性（MRH）域。另外，受体的二聚化可能在膜上发生，使其表征具有挑战性。在这项研究中，最初用于hM6PR细胞摄取测定的新型人M6PR（hM6PR）过表达细胞系用于生产hM6PR-ECD，并开发了新型小分子仿生（氨基苯基-M6P）亲和树脂用于纯化M6PR-ECD。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和基于表面等离振子共振的结合试验中，证明其配体结合功能，