Identification of residues essential for carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor
Identification of residues essential for carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor
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DOI：
10.1074/jbc.m109855200
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发表时间：
2002-03-29
影响因子：
4.8
通讯作者：
Dahms, NM
中科院分区：
文献类型：
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作者：
Hancock, MK;Haskins, DJ;Dahms, NM
两种不同的甘露糖6-磷酸（Man-6-P）受体（MPR），阳离子依赖性MPR （CD-MPR）和胰岛素样生长因子II/MPR (IGF-II/MPR)，可识别不同的Man-6-P配体。IGF-II/MPR是一种I型跨膜糖蛋白，具有由15个重复结构域组成的大胞质区，这些重复结构域彼此之间和CD-MPR的单个胞质结构域具有序列同一性。IGF-II/MPR的两个不同的Man-6-P结合位点与CD-MPR的基于结构的序列比对表明，IGF-II/MPR结构域3和9的几个残基是Man-6-P结合所必需的。为了验证这一假设，在编码牛IGF-II/ MPR的N端或c端man -6- p结合位点的构建体中进行了单氨基酸替换。利用磷酸五氨基糖-琼脂糖亲和层析分析了COS-1细胞分泌的突变体IGF-II/ mpr，鉴定出结构域3的4个残基（Gln-392、Ser-431、Glu-460和tir -465）和结构域9的4个残基（Gln-1292、hs -1329、Glu-1354和tir -1360）是识别Man-6-P所必需的。利用溶酶体酶-葡萄糖醛酸酶的结合亲和力研究证实了这些结果。总之，这些分析提供了强有力的证据，证明IGF-II/MPR的两个man -6- p结合位点在结构上彼此相似，与CD-MPR相似，并利用相似的碳水化合物识别机制。
Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/ MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N- or the C-terminal Man-6-P-binding sites of the bovine IGF-II/ MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphate-agarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, beta-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.