Mannose 6-Phosphate Receptor-mediated Uptake Is Defective in Acid Sphingomyelinase-deficient Macrophages: IMPLICATIONS FOR NIEMANN-PICK DISEASE ENZYME REPLACEMENT THERAPY

Rajwinder Dhami,Edward H Schuchman

doi:10.1074/jbc.m309465200

Rajwinder Dhami, Edward H Schuchman

Open Access

https://doi.org/10.1074/jbc.m309465200

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Abstract

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 approximately 10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.

Highlights

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 elucidate which receptors were important for uptake of rhASM by alveolar macrophages, cells were incubated with rhASM alone, or in the presence of excess mannan or mannose 6-phosphate (M6P) (Fig. 1D)
In ASM knock-out (ASMKO) macrophage cultures mannan inhibited uptake of rhASM by ϳ60%, but M6P did not affect rhASM uptake at all. These results suggested that M6PRs were either not present on the ASMKO cell surface or were not functional

Summary

Introduction

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). We 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 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 ϳ10-fold greater when compared with the “complex” type rhASM. These findings have important implications for NPD enzyme replacement therapy, in the lung. The MR is a pattern recognition receptor for various microorganisms and has been shown to play an important role in host defense and scavenger function [22], whereas the primary function of the M6PR appears to be the recapture of secreted lysosomal hydrolases and transport to the lyso-

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