Mannose 6-phosphate receptor-mediated uptake is defective in acid sphingomyelinase-deficient macrophages - Implications for Niemann-Pick disease enzyme replacement therapy
Mannose 6-phosphate receptor-mediated uptake is defective in acid sphingomyelinase-deficient macrophages - Implications for Niemann-Pick disease enzyme replacement therapy
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DOI：
10.1074/jbc.m309465200
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发表时间：
2004-01-09
影响因子：
4.8
通讯作者：
Schuchman, EH
中科院分区：
文献类型：
--
作者：
Dhami, R;Schuchman, EH
富含脂质的巨噬细胞进行性积聚是酸性鞘磷脂酶(ASM)缺陷型Niemann-Pick病(即A型和B型NPD)的特征。为了探讨酶替代疗法治疗这种疾病的机制，我们研究了ASM基因敲除(ASMKO)小鼠肺泡巨噬细胞对重组人ASM(RhASM)的摄取。用于这些研究的重组酶是在中国仓鼠卵巢细胞中产生的，含有复合型N-连接寡糖。与正常巨噬细胞相比，放射性标记的rhASM与ASMKO巨噬细胞的结合增强，这与其更大的体积和更大的表面积是一致的。然而，与正常细胞相比，ASMKO细胞对该酶的内化明显减少。用受体特异性配体抑制酶摄取的研究表明，在正常细胞中，rhASM被甘露糖和甘露糖6-磷酸受体(分别为MR和M6PR)所摄取，而在ASMKO细胞中，M6PR对rhASM摄取的作用很小。M6PR mRNA在ASMKO细胞中的表达正常，但Western blotting显示这些细胞中的受体比正常细胞多。因此，我们假设ASMKO巨噬细胞中的脂质积累导致M6PR运输和/或降解异常，导致酶摄取减少。与这一假设一致，我们还发现，当rhASM被修饰以暴露末端甘露糖残基和靶向甘露糖受体时，ASMKO细胞对这种修饰后的酶形式的摄取类似于“复合型”rhASM的10倍。这些发现对NPD酶替代疗法有重要意义，特别是在肺部。
Progressive accumulation of lipid-laden macrophages is a hallmark of the acid sphingomyelinase (ASM)-deficient forms of Niemann-Pick disease (i.e. Types A and B NPD). To investigate the mechanisms underlying enzyme replacement therapy for this disorder, we studied the uptake of recombinant, human ASM (rhASM) by alveolar macrophages from ASM knock-out (ASMKO) mice. The recombinant enzyme used for these studies was produced in Chinese hamster ovary cells and contained complex type, N-linked oligosaccharides. Binding of radiolabeled, rhASM to the ASMKO macrophages was enhanced as compared with normal macrophages, consistent with their larger size and increased surface area. However, internalization of the enzyme by the ASMKO cells was markedly reduced when compared with normal cells. Studies using receptor-specific ligands to inhibit enzyme uptake revealed that in normal cells rhASM was taken up by a combination of mannose and mannose 6-phosphate receptors (MR and M6PR, respectively), whereas in the ASMKO cells the M6PR had a minimal role in rhASM uptake. Expression of M6PR mRNA was normal in the ASMKO cells, although Western blotting revealed more receptors in these cells when compared with normal. We therefore hypothesized that lipid accumulation in ASMKO macrophages led to abnormalities in M6PR trafficking and/or degradation, resulting in reduced enzyme uptake. Consistent with this hypothesis, we also found that, when rhASM was modified to expose terminal mannose residues and target mannose receptors, the uptake of this modified enzyme form by ASMKO cells was similar to10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.