Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (Mannose 6-phosphate n-acetylglucosamine ester)
Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (Mannose 6-phosphate n-acetylglucosamine ester)
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DOI：
10.1021/bi7011806
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发表时间：
2007-11-06
期刊：
影响因子：
2.9
通讯作者：
Dahms, Nancy M.
中科院分区：
文献类型：
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作者：
Chavez, Carrie A.;Bohnsack, Richard N.;Dahms, Nancy M.
300 kDa 阳离子非依赖性甘露糖 6-磷酸受体 (CI-MPR) 和 46 kDa 阳离子依赖性 MPR (CD-MPR) 是溶酶体酶靶向系统的关键组成部分，该系统结合新合成的含 6-磷酸甘露糖 (Man-6-P) 的酸性水解酶，并将其从分泌途径转移。先前的研究已将 CI-MPR 的两个高亲和力 Man-6-P 结合位点映射到结构域 1-3 和 9，并将一个低亲和力位点映射到其 15 个结构域胞质外区域内的结构域 5。基于结构的序列比对预测结构域 5 包含四个保守残基（Gln、Arg、Glu、Tyr），这些残基被确定为 CD-MPR 和 CI-MPR 结构域 1-3 和 9 结合 Man-6-P 所必需的。在这里，我们通过对含有单个氨基酸取代的构建体进行表面等离子共振（SPR）分析表明，这些保守残基（Gln-644、Arg-687、Glu-709、Tyr-714）对于结构域5的碳水化合物识别至关重要。此外，结构域5的位置711处的N-糖基化位点（预计位于结合口袋附近）对碳水化合物结合亲和力没有影响。仅含有磷酸单酯 (Man-6-P) 或磷酸二酯 (甘露糖 6-磷酸 N-乙酰氨基葡萄糖酯, Man-P-GlcNAc) 的 MPR 内源配体是通过用重组 GlcNAc-磷酸转移酶和揭示酶 (N-乙酰氨基葡萄糖-1-磷酸二酯) 处理溶酶体酶酸性 α-葡萄糖苷酶 (GAA) 生成的α-N-乙酰葡糖胺酶）。使用这些修饰的 GAA 进行的 SPR 分析表明，与 CD-MPR 或 CI-MPR 的结构域 9 不同，结构域 5 对 Man-P-GlcNAc 的亲和力比 Man-6-P 高 14-18 倍，表明该受体区域将含磷酸二酯的溶酶体酶靶向至溶酶体。
The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosarninidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.